Globular proteins at solid/liquid interfaces

517 C&x& nnrf Surfucrs B, Biornrerfaces. 2 ( 1994) 517-566 0927-7763 9.i 907.00 8 1994 - Elsevier Sctcnce B.V. All nghts reserved.

Review

(Received 18 May 1993; accepted 19 October 1993)

Abstract

Seven years have passed since one of us (W.N.) published the last comprehensive review on the mechanism of globular protein adsorptron to solid/water interfaces. Since that time, annual contrtbutions to the field have steadily increased and substantial progress has been made in a number of important areas. Thts review takes a fresh look at the driving force for protein adsorption by combinrng recent advances wtth key results from the past. The analysts indicates that four e&cts. namely structural rearrangements in the protein molecule. dehydration of (parts of) the sorbent surface, redistrtbution of charged groups in the interfacial layer, and protein surface polarity usually make the primary contributions to the overall adsorption behavior.

Ke_r words: Adsorption behavior; Globular proteins; Sohd,iliquid Interfaces

1. rntro~uction

Exposure of an aqueous protein solution to a solid surface typically results in the excess accumulation of protein molecules at the solid/liquid interface. This tendency for proteins to adsorb spontaneously is now thought important in a variety of natural and synthetic processes. As a result, protein adsorption is attracting attention in research disciplines ranging from geophysics and materials science to biomedical engineering and optometry~ For instance, evolutionary scientists now believe that the enrichment of proteinaceous compounds at soil surfaces played an essential role in the prebiotic stage of the genesis of terrestrial life Cl,23 possibly through the entrapment and stabilization of crude secondary structures [ 31. Materials and food scientists continue to discover potent uses for proteins in the stabilization of microemulsions, pharmaceutical creams and

*Corresponding author.

lotions, formulated foods. and foams such as those used in oil spill removal and in the manufacture of light-weight, high-tensile-strength foamed con- cretes [4,.5].

In the medical sciences. pathologists and biomedical engineers have determined that thrombus development on cardiovascular implant materials is intimately related to protein adsorption processes involving fibrinogen. Factor 11 and Factor 12. high molecular weight kininogen, and possibly a number of other plasma proteins [S-SO]. Similar phenomena are involved in blood coagulation. complement activation, artificial kidney failure, plaque formation on teeth and dental restoratives [ I1,12], and the fouling of contact lenses by tear proteins [ 13-161. These undesirable medical effects are contrasted by recent applications of controlled protein adsorption in the development of drug delivery systems [17] and biosensors for in vivo monitoring of glucose levels in blood and for in vitro immunoassays and other serological tests [18].

51s C A. Huyws and Ctt Sorde.‘Collords Surfaces B. Bminterfaces I ( 1994) 517-566

Protein adsorption research in the life sciences has arisen naturally from the in vivo tendency of many proteins to locate and function at a phase boundary. Pancreatic lipases which control the digestion of alimentary fats in the duodenum function at an oil/water interface [19-201. A large class of proteins is intercalated into lipid bilayers, or biological membranes, which create a suitable environment for the action of these proteins. Membrane proteins serve as pumps, receptors, gates, energy transducers and enzymes. For instance, photosynthesis occurs in the inner membrane of chloroplasts, and oxidative phosphoryla- tion, in which adenosine triphosphate (ATP) is formed by the oxidation of fuel molecules, takes place in the inner membranes of mitochondria [21,22].

Protein adsorption is also a boon and bane to modern technology, particularly biotechnology. Controlled and/or well-characterized protein adsorption at solid/liquid interfaces is an important goal in much of the current research directed towards immobilized-enzyme bioreactors and protein purification systems. For instance, many chro- matographic separations, such as hydrophobic, displacement and ion-exchange chromatographies, are based on differences in binding affinities of proteins for the support material. High density in vitro cell cultures typically require cell-surface adhesion which is mediated by a sublayer of adsorbed proteins [ 23,241. Negative consequences of protein adsorption processes include the highly undesirable fouling of heat exchangers, ultrafiltra- tion membranes, sea-water desalination units, ship hulls, food processing units, etc. [25].

An important result of this rich and diverse research effort has been the steady accumulation of new experimental and theoretical strategies for studying protein adsorption. Research on the kinetics of protein adsorption, which is essential for understanding and controlling dynamic processes such as blood coagulation and membrane fouling, has motivated the development of a number of in situ methods for determining rates of adsorption and adsorbed amounts: examples include total

internal reflection fluorescence (TIRF), reflection infrared spectroscopy. and ellipsometry. Experiments to determine conformations and activities of adsorbed proteins have involved novel applications of NMR spectroscopy, Raman and IR spectroscopies, fluorescence probes, calorime-

try, transmission circular dichroism, etc. [ 1326-321. Clearly, no single experimental or theoretical approach can answer all questions concerning protein adsorption. Instead, meaningful conclusions must be drawn from a synthesis of results from various disciplines, where the focus of the research is often such that only certain aspects of the protein adsorption problem can or need be addressed. Such an approach has already resulted in the discovery and general acceptance of a number of physical and energetic properties which are common to most, if not all, protein adsorption systems. However, many important questions remain unanswered, and a unified predictive theory is not within sight.

This review focuses on thermodynamic (including electrostatic) aspects of protein adsorption with the aim of indicating some general principles and resolving the dominant forces governing the adsorption process. Attention will be focused on the adsorption of globular proteins from aqueous solution onto solid non-porous surfaces. No attempt has been made to provide an exhaustive survey of the literature. Instead, we focus on those studies where the protein and the surface have been sufficiently characterized to permit the formulation of meaningful, unambiguous conclusions concerning the driving force for adsorption. Regrettably, few such studies have appeared in the literature, partly because an understanding of the thermodynamics of protein adsorption is a compo- nent but not the focus of much of the research on proteins at interfaces. For instance, the extensive literature on the mechanism of thrombosis is pri- marily concerned with the competitive adsorption of blood proteins from complex multicomponent solutions onto implant materials. Here, important information has been obtained concerning the time evolution and bulk-concentration dependence of

C.d. H~~~nrs cd FV .Vorde;Colloids Swfuces B: Biorntrrfuces 2 ( 19Y1 J 517-566 519

the surface concentration and composition, but the complexity of the system prevents one from gaining quantitative (or often even qualitative) insights into the thermodynamic driving forces for adsorption.

Regardless of the mechanism and kinetics of the process, protein adsorption (at constant temperature and pressure) can only occur if the Gibbs energy G of the system decreases:

A,*,G = AadsH - TA,,,S < 0 (1)

where H, S and T are the enthalpy, entropy and absolute temperature respectively, and Aads indicates the change in the thermodynamic functions of state resulting from the adsorption process. An answer to the question why proteins prefer interfaces may thus be found by considering which interactions contribute to AadsG.

Protein adsorption is the net result of the various interactions between and within the system components, which include the sorbent surface, the protein molecules, the solvent (water) and any other solutes present such as low molecular mass ions [33]. The origins of these interactions include Lifshitz-van der Waals forces (i.e. dispersion, orientation and induction forces), Lewis acid-base forces (including hydrogen bond forces), electrostatic forces (including ion pairing) and more entropi- cally based effects such as the hydrophobic effect (at least under ambient conditions) and internal packing (steric/excluded-volume) restrictions. Clearly, intermolecular interactions between, for example, protein and surface, solvent and surface, or solvent and solvent are important in protein adsorption. In addition, intramolecular forces between atoms and residues within the protein macromolecule may contribute to AadeG. Thus a complete understanding of the driving force for protein adsorption requires a general knowledge of structures and stabilities of globular proteins.

2. Protein structures

Proteins are copolymers of some 20-23 different L-amino acids which are linked to each other to form a linear polypeptide chain (see Fig. 1). Many

Fig. 1. Structure of a peptlde unit in a polypeptide cham. Two (4 and (p) of the three (backbone) bonds are free to rotate; the

shaded bond is fixed. R and R’ represent amino acid side-groups.

proteins consist of a single polypeptide chain; others contain two or more chains which may be either identical or different. Two ($ and 4) of the bonds in the peptide unit are. in principle, free to rotate; the C-N bond, which is shaded in Fig. 1, is fixed because of its partial double-bond character induced by mesomery. The side-chains R, R’, . . .

vary in size, shape, charge and hydrogen bonding capacity. Some of the side-chains are acidic; others are basic, which renders the polypeptide ampho- teric. Side-chains also vary in hydrophobicity (polarity), making the polypeptide chain amphiphi- lit as well. It is therefore not surprising that most proteins are highly surface active.

A protein molecule’s primary structure, which is the sequence of amino acids and the location of any disulfide bridges in the polypeptide chain, ultimately determines the spatial organization of the protein molecule in a given environment. For single-strand proteins, this spatial organization is often divided into two categories: secondary structure, which is any regular local structure (e.g. r- helix, p-pleated sheet) in a linear segment of the polypeptide chain, and tertiary structure, which is the overall topology of the polypeptide chain.

Three-dimensional structures of proteins may be broadly classified into (a) molecules that are highly solvated and flexible. resulting in an expanded (but

520 CA. Hawes and W ,Vorde Colloids Surfaces B Blomterfaces 2 (lY94) jli’-566

rarely random) coil structure, (b) molecules that have adopted a regular structure like an r-helix (Fig. 2(a)) or a p-pleated sheet (Fig. 2(b)), the fibrous proteins, and (c) densely packed molecules of roughly spherical shape containing a consider- able amount and variety of internal architecture, the globular proteins (e.g. enzymes, immunological proteins and transport proteins).

In the expanded-coil structure, which many proteins appear to assume in the unfolded or denatured state, the polypeptide chain can attain numerous conformations because of the rotational freedom of the $ and Q bonds along the main chain. However, the bulkiness of the side-groups imposes sterical restrictions on these rotations such that only a limited number of combinations of rotations are allowed. For instance, amino acid side-chains are almost always positioned in the trans configuration to minimize steric hindrance. A detailed analysis of the possible values of $ and

d was first made by Ramachandran and Sasisekharan [34] using hard-sphere models for the atoms and a fixed geometry for the bonds. The resulting Ramachandran plots suggest that the number of distinct conformations (excluding side- group conformations) in a-helix and P-sheet struc-

(a) (b)

Fig. 2. Ordered secondary structures in polypeptide chains: (a) x-helix; (b) parallel p-pleated sheet.

tures is about one-fourth the number of totally available conformations in a random polypeptide structure [35]. For proteins belonging to classes (b) and (c) mentioned above, the conformation of

the polypeptide backbone is more or less fixed. For instance, native states of globular proteins are extremely compact; their free volumes, compress-

ibilities, and conformational freedom are comparable to those in glasses and polymer crystals [36]. Such a compact inflexible architecture is possible

only if interactions within the protein molecule

and between the protein molecule and its environ-

ment are sufficiently favorable to. compensate for the substantial loss of conformational entropy.

Globular proteins in an aqueous environment have a number of structural characteristics in

common [35]. ( 1) They are more or less spherical, with molecu-

lar dimensions in the range of a few to a few tens

of nanometers. (2) Hydrophobic side-groups tend to be buried

in the interior of the molecule where they are shielded from contact with water. As a result, part of the hydrophilic hydrogen-bond-forming polypeptide backbone must also locate in the interior. Therefore one important property of secondary

structures such as cc-helices and P-sheets is the efficient matching of hydrogen-bond donors and acceptors between internal polar groups of the

polypeptide backbone. (3) Charged groups are almost always located

in the aqueous periphery of the protein. Any

charged groups in the interior occur as ion pairs since dissociation is strongly opposed by the low

local dielectric permittivity. (4) The atoms are densely packed, with most

adjacent atoms in van der Waals contact. Internal atomic packing densities average 75%, which is similar to the maximum packing density of equal- sized hard spheres. For comparison, water and cyclohexane have packing densities of 58% and

44% respectively, at 25 ‘C and 1 atm. Figures 3(a)-3(c) are computer graphics images

of native bovine pancreas ribonuclease (RNase)

C.A. Haynes and W. Norde/Colloids Surfaces B: Biointerfaces 2 (1994) 517-566 521

Fig. 3. Molecular graphics images of bovine pancreas ribonuclease (RNase). (a) Polypeptide backbone showmg a-hehx (orange), /?-sheets (blue) and randomly structured parts. (b) Dtstribution of proton tttratable groups at the protem surface. Color scheme accordmg to increasing order of pK value: dark red, terminal carboxyl group; light red, asparttc and glutamtc carboxyl groups; magenta, htsttdine; cyan, termmal amino

obtained using the Silicon Graphics Iris 4D/70GT workstation, Biosym’s INSIGHT software, and

atomic coordinates provided by the Protein Data Bank (Brookhaven National Laboratory). Figure 3(a) shows the folding pattern of the poly-

peptide backbone, Fig. 3(b) shows the charged groups at the protein surface, and Fig. 3(c) shows

the hydrophobicity of the protein exterior using a color scheme based on Eisenberg’s atomic solvation parameter Ag/A,, where Ag is the Gibbs energy change when transferring the atom from

n-octanol to water and A, is the water-accessible

surface area of the atom [37]. A couple of structural characteristics, beyond those listed above, are apparent in these images. First, not all hydrophobic residues are hidden in the interior. Apolar atoms occupy between 40% and 50% of the water- accessible surface area of most small (or asymmetric) proteins such as RNase (molecular weight

(MW) 13 680 Da), giving them relatively high surface hydrophobicities. For larger (or more spherical) proteins, which possess lower surface

area/volume ratios, the percentage of apolar atoms

on the surface is typically less. The apolar content of the interior of RNase is about 60%, which is typical of most small globular proteins [ 381. Secondly, as shown in Fig. 3(c), polar and apolar residues are more or less evenly distributed over the protein surface; no regions are observed where

the surface shows either a distinctly hydrophilic or hydrophobic character.

group; light blue, lysine amino group; dark blue, guamdyl group. (c) Distribution of polar and apolar groups at the protein surface. Color scheme based on Etsenberg’s atonuc solvation parameter (see text):

Atom Color

C N, 0 O- N+

+21+ 10 +16+2

-6i4 -24&10 -50+9

Yellow Yellow Magenta Light blue Dark blue

3. Factors affecting protein folding and stability

3.1. Doininunt i$ects: conforrn~t~o~~~l free~~orn and

~ydro~~zob~c dehydration

For most globular proteins, 40-70% of the polypeptide chain participates in either an a-helix or a P-sheet. Both of these secondary structures are characterized by ~lydrogeil-bond forination between peptide units which reduces the rotational mobility of the polypeptide chain and hence the conformational entropy. Assuming four possible conformations per peptide unit in the expanded- coil structure and only one in the cr-helix or p- sheet, the loss in conformational entropy per peptide unit is R In (l/4)= - 11.53 J K-’ mol-‘. Thus a large entropy decrease (-- 577 J K- ’ per mole protein), equating to a Gibbs energy increase of 173.0 kJ rn01-~ at 300 K, will result from the folding of a protein consisting of 100 amino acids into a structure where 50% of the residues are involved in a-helices or /?-sheets. Additional losses in conformational entropy will result from the “freezmg” of other parts of the polypeptide backbone (and possibly some side-chains) into densely packed random structures within the interior of the protein.

Protein folding requires that the large conformational entropy opposing the folded state be out- weighed by the sum of the enthalpic forces and other entropic forces affecting stability. Although a number of interactions are known to affect stability, there is now strong evidence that one type of interaction, which Tanford termed the “llydrophobic effect” [39], provides the dolniilallt driving force for the folding of globular proteins in water [36,40-461. The term “hydrophobic effect” originates from the observation that the solubilities of small non-polar solutes in water are extremely low and exhibit minima at intermediate teii~peratures~ moreover, addition of non-polar solutes to water creates a large positive change in the heat capacity of the aqueous solution [39]. This behavior is in sharp contrast with that of “regular solutions” (which include most non-

aqueous mixtures), where the enthalpy and entropy of mixing show little or no temperature dependence and the Gibbs energy (i.e. the solubility) is linearly dependent on temperature [4’7,48]. Recently, Privalov and Gill [46] used micro-differential- scanning calorimetry (micro-DSC) to determine the temperature dependence of the Gibbs energy, enthalpy and entropy changes associated with transferring a mole of non-polar solute (benzene) into an excess of pure water (see Fig. 4). The immiscibility of benzene and water is reflected in the always positive value of AG. Of course, such immiscibility is not restricted to mixtures of non- polar solutes in water. The signature of the hydrophobic effect is the strong temperature dependence of AN and AS which causes AG to achieve a maximum value and the solubility to reach a mimmum at an intermediate temperature (where the solubility is determined oniy by enthalpic effects).

In regular solutions, immiscibility is the result of a positive enthalpy of transfer. In contrast, the immiscibility of benzene in water at ambient temperatures is caused by a large negative entropy of

-i 5 E

350 400 450

temperature I K

FIN 4. Gibbs energy (AC), enthalpy (AH) and entropy (AS) of transferring benzene from a pure benzene phase ml0 m aqueous solutmn. Data from Pr~valov and Gill [46], reprInted wth permission.

C..-l. Haxnes und Ct: ;Lbrde, Colioids Surjucrs B. Bwintrrfuws 2 ( 1 YYf ) 5 17-jljfi

transfer. The molecular picture consistent with this observation is a non-polar solute molecule with

water molecules highly oriented around it so as to

form the maximum number of hydrogen bonds, none of which can be formed with the solute. As

the temperature is increased, this low-entropy solvent configuration is no longer favored, and the ordered water molecules will “melt” away from the solute surface back into the bulk solution. This melting process provides an additional energy- storage mechanism for the solvent which gives rise

to the large heat capacity increase upon mixing [49]. At high temperatures, it is the now positive enthalpy of transfer which prevents miscibility. At

all temperatures, non-polar groups are rejected from the aqueous environment rather than being (intramolecularly) attracted to other non-polar

groups. The importance of the hydrophobic effect in

protein folding and stability was first recognized by Kauzmann [40], who reasoned that protein folding is driven by a large entropy gain in the

solvent molecules released from hydrophobic residues during the folding process [41]. Thus the existence of compact globuiar proteins in solution is an illustration of the constructive power of chaos:

the ordered protein structure is preferred because it corresponds to an increased disorder in the surrounding water.

Indirect experimental confirmation of Kauzmann’s theory was achieved by Privalov and

Khechinashvili [ 50 J who used micro-DSC, isothermal microcalorimetry and proton titrations to

determine enthalpies, entropies and Gibbs energies

of denaturation (e.g. A&nG = Gdenaturcd Sfate - G rolded ,& for small single-domain proteins as a function of temperature and pH. For two single- domain proteins, hen egg-white lysozyme and calcium-containing r-lactalbumin from bovine mifk, Fig, 5 shows denaturation energies and entropies obtained by Haynes et al. [Sl] using the procedure of Privalov [52 J. Here, AN_&. for example, is the total enthalpy change resulting

from the denaturation process and therefore

a 20 LO 60 60 100

ttmperature i t

0 20 40 60 80 100 temperature /‘C

Fig. 5. D~naturatlon enthatpies (A,&), entropies (A,& and Gibbs energies (A,oG) for native (a) hen egg-white lysozyme and (b) calcium-containmg z-lactalbumm in 50 mM KC1 solution. Data from Haynes and Norde [26].

includes a number of effects in addition to hydrophobic dehydration. Nevertheless, the basic features of the hydrophobic effect are evident; AN-&Y and TA.,_,S are strongly temperature dependent, resulting in a pronounced non-linearity in A>_nG(T).

A second equally important observation can be drawn from Fig. 5. Even under optimum solution conditions, the folded conformation of a globular protein is only marginally stable. For singie- domain proteins, maximum values for AN_nG (i.e. maximum native-state stabilities) fall in the range 20-100 kJ mol-‘, roughly equivalent to the energy required to rupture 1-8 hydrogen bonds. This indicates that protein unfolding is a highly cooperative process; the disruption of any significant portion of the folded structure leads to unfolding of all the rest. Thus, although conformational- entropy and hydrophobic effects dominate the folding process, no type of molecular interaction is unimportant (see Table If.

Table 1 Interacttons determinmg the three-dimensional structures of proteins dtssolved in aqueous rolutton

Type of mternctton Conttibutton Remarks to Art, G

Conformational entropy

Hydrophobtc dehydration

Hydrogen bond and dipole

Coulomb

Dispersion

Dtstorttons of bond lengths and bond angles

>o (‘?)

> or co

Folding, especially the formation of ordered secondary structures such as I- helix or P-sheets, reduces the conformational entropy of the polypeptide chain

Large entropy increase in water molecules released from contact with hydrophobic residues

Formatton of protein-protein and water-water bonds compensated by loss of (more favorable) protein-water bonds

Dependmg on the pH relative to the isoelectrtc pomt of the protein’sorbent complex

Slightly favor the folded structure since atomic packing densities are extremely htgh in folded proteins

Some bonds are under stress in the compact folded structure

3.2. Hydrogen bonds

Each peptide unit (except Pro) in a protein’s backbone contains a hydrogen donor (>NH) and a strong proton acceptor (>C=O), the basic con- stituents of a hydrogen bond. For a series of globular proteins, Baker and Hubbard [53] found that 88% of all peptide amide groups and 89% of all peptide carbonyl groups are involved in hydrogen bonds. Some amino acid side-chains can participate in hydrogen bonds as well, but their contribution to the total number of internal hydrogen-bonds is typically small. A statistical breakdown of all hydrogen bonds involving peptide amides reveaIed that 68% are to backbone carbony groups, 11% are to side-chains and 21% are to water; for main-chain >C=O groups, the percentages are 46% to backbone amides, 11% to side-chains, and 43% to water [53]. Moreover, the

0 unique 3.6 residues, 5.41 A translation per turn structure of the r-helix (Fig. 2(a)) is due in large part to the strong hydrogen bonds formed between i carbonyl oxygen atoms and i +4 amide groups of the polypeptide backbone; similar arguments apply to parallel and antiparallel P-sheet structures. Clearly, hydrogen bonds make important contributions to secondary and tertiary structures of gfobu- lar proteins.

The influence of intrachain hydrogen bonds on protein stability remains unclear despite substantial attention given to the subject over the past 30 years [36,41,54]. Creighton (Ref. 35, p. 327) suggests that hydrogen bonds contribute between 200 and 3000 kJ mol-’ to the stability of the folded conformation. He argues that intrachain hydrogen bonds, which are linear (and therefore strong) and present essentially all the time, are energetically more favorable than water-water or water-peptide hydrogen bonds, which are present only about half the time at room temperature and are frequently distorted. However, experiments on model compounds do not support this view. For instance, an elegant series of studies by Klotz and co-workers [55,56] using ~-methylacetamide dimerization in water and in Ccl, as a model for peptide-water and peptide-peptide hydrogen bonds. respectively, revealed that amide-water hydrogen-bonds are favored over intrachain hydrogen-bonds; the results of Klotz and co-workers suggest that hydrogen-bond forces destabilize the folded conformations of globular proteins.

Another possibly more realistic approach to establishing the contribution of hydrogen bonds to folded-state stabilities follows from studies of helix-coil transitions in polyamino acids. Table 2 presents thermodynamic data for the helix-to-coil

CA. Hayes and Ct: NordeiCollolds Surfaces B: Biointeqaces 2 ( 1994) 517-556 53

Table 2 Thermodynamic changes of state (kJ per mole peptide unit) for helix-to-coil transitions in poly(amino acid)s in aqueous solution at 25’C

Poly(amino acid)

Poly(L-glutamate) Poly(L-leucine)

A&G A&H TA&

0.58 4.3s 3.80 3.50 3.50 0

transitions of poly (L-glutamate) and poly (L-leu- tine) in aqueous solution at 25°C [ 161. For both polymers, the Gibbs energy change A,,,G is positive for the helix-to-coil transition, indicating that the helix is thermodynamically favored under the solution conditions. This, however, does not prove that hydrogen-bond forces stabilize the folded conformation; other factors, particularly changes in hydration of apolar side-groups and in rotational mobility along the polypeptide backbone, also contribute to A,,+G. If hydrophobic effects are present, the ordering of water molecules around hydrophobic groups during the helix-to-coil transition should create a large positive change in heat capacity (see Section 3.1.). Such a change has indeed been observed for the poly (L-leucine) system. Moreover, the absence of an entropic contribution to As-G for this system indicates that the hydration effect is large enough to nullify the conformational entropy gain in the (unfolded) polypeptide backbone. For the more polar poly (L-

glutamate), the heat capacity change is essentially zero, suggesting that hydrophobic effects are not involved in the transition. Here, hydrogen-bond forces and backbone rotational mobility changes are likely to make the only significant contributions to the transition energy and entropy. In a helix, all bond angles along the polypeptide chain are essentially fixed. In the random-coil structure, two of the three bonds of the peptide unit are free to rotate within the constraints imposed by any additional restrictions, such as steric hindrance by the side-groups. Gaining one degree of rotational freedom involves an enthalpy and entropy increase of RT and R In 2 respectively. Each peptide unit is capable of gaining up to two degrees of freedom.

Taking this into consideration, we can estimate the contribution of intrachain hydrogen bonds to the helix-to-coil transition as a function of the increase in backbone rotational freedom per peptide unit (see Fig. 6). If the change in rotational mobility is small, hydrogen bonds appear to favor the helix

(i.e. Ai,-cG~-bond >O); helix destabilization is predicted for larger, possibly more realistic changes in rotational mobility. However, Ah-cGH_bond is always small, suggesting that intrachain hydrogen bonds do not make a large contribution to globular protein stabilities. Indeed, the large difference in the values of AhXG for the poly (L-leucine) and poly (L-glutamate) systems indicates that hydrophobic dehydration probably drives the collapse of random hydrophobic poly(amino acid) (i.e. protein) coils in aqueous solution.

3.3. Lifshitz-van der Waals interactions

Lifshitz-van der Waals attractions arise from interactions between fixed or induced dipoles. They are very sensitive to the distance I between atoms, varying as r -6. Since atomic packing densities are unusually large in the interiors of globular proteins, van der Waals attractions should favor the folded conformation. However, their overall effect on protein stabilities remains unclear. Difficulties in assessing the contribution of Lifshitz-van der

Fig. 6. Thermodynamic data at 25’C for hydrogen-bond formation in poly(L-glutamate) as a function of the rotational freedom per bond in the polypeptide chain. There are two bonds which are free to rotate in the polypeptide chain.

Waals attractions to protein folding lie primariliy in the lack of simple macroscopic tests which can distinguish them from hydrogen-bond forces despite the obvious microscopic differences between these two interactions [36]. Despite these uncertainties. Lifshitz-van der Waals attractions are likely to make significant contributions to the stability of the folded state as evidenced by the differences between Hamaker constants for globular proteins and pure water [.57,58].

3.4. Bond lengths and angles

Distortions of optimum bond lengths and bond angles within a protein are likely to be negligible in the loose, low-segment-density denatured state. However, such distortions may occur in the folded state, particularly within the tightly packed interior. Energy minimization calculations indicate that covalent bond distortions in native (crystallo- graphic) proteins provide a small but significant opposition to the folded state stability [35,59], averaging 4-8 kJ per mole of peptide unit. Apparently, minor bond distortions are necessary to achieve a tightly packed, predominantly apolar interior where dispersion forces and peptide-peptide hydrogen bonds are at or near an optimum.

3.5. Coulomb interactions

A semiquantitative understanding of the effect coulomb interactions between charged surface groups has on folded protein stabilities is provided by Tanford and Kirkwood’s model [60-621 for calculating the electrostatic Gibbs energy G,, of a spherical particle (radius 1 nm; EE~=~, where ecO is the dielectric permittivity of the region and E is the dielectric constant) bearing discrete charges on its surface. The energy G,, represents the reversible work required to place all of the discrete (point) charges on the originally uncharged particle at constant temperature and pressure:

G,, = 1 4 dQ q=o

where 4 is the electrostatic potential and Q is the proton charge of the particle. Using an approximate expression for 4(Q) derived by Kirkwood [ 631. Tanford [ 641 solved Eq. (2) for the “discrete- charge” model shown in Fig. 7 (see Table 3). In the isoelectric region, coulomb attractions between oppositely charged residues on the “protein” surface favor (G,, ~0) a compact folded conformation (assuming the positive and negative charges are evenly distributed over the surface). However, an excess of positive or negative surface charge readily leads to charge-charge repulsion and thus promotes a more expanded structure since the charge density on the folded molecule is greater than on the unfolded molecule. As expected. the dependence of G,, on solution pH is reduced at higher ionic strengths since salts shield electrostatic forces.

Specific interactions. such as ion pairs between

/ N/cAy \ \ /I

C ‘lr-- --N\ \

\ / \ : \ \ \

,& qQ/J

Fig. 7. Model “protein” used for calculating the electrostatic

Gibbs energies shown in Table 3. C. N and I represent carboxyl,

amino and imidazole groups respectively. Reprinted from

Tanford [64] with permission.

Table 3

Electrostatic Gibbs energy G,, of the model “protein” shown tn

Fig. 7 as a function of protein net charge and ionic strength I

Net charge

z+ G., (kJ mol-‘)

I=0 f=O.O15 I = 0.060

+6 +31.5 + 24.6 f21.4

+4 + 1.59 -0.13 -0.71

+2 - 17.5 - 16.2 -15.1

0 -26.7 - 23.6 -21.7 -I

1; - + 17.0 4.60 - +2.93 15.3 - + lx! 2.30

CA. Haxnes and W. Xmie:Colloids Surfaces B: Bwinterfaces 2 f 19941 Sf 7-566

oppositely charged residues in close spatial proxim- establish relative hydrophobicities of solid (s) sur- ity, can also exert a modest influence on protein faces; most common are the contact angle 6 of a stability. In natural proteins. ion pairs are found sessile drop of pure water (w), the Gibbs energy almost exclusively on or near the protein surface (or reversible work) of hydration, AG,,, and the and tend to stabilize the compact folded conforma- pure-solid surface tension yJ. For reversible wetting tion [6.5]. For example, the Asp 70-His 31 ion at constant temperature and pressure, these pair in T4 lysozyme stabilizes the native conforma- thermodynamic quantities are related by Young’s tion between 12 and 20 kJ mol-’ [66]. Law (see Fig. 8):

All proteins become highly unstable or denature at extreme pH values. Much of this is due to the global electrostatic repulsion created by the high surface charge density. An additional driving force for denaturation may arise from the ionization of residues originally buried in the folded protein in the non-ionized form. For instance, the non-ionized forms of His and Tyr residues, which are commonly found in the interior of globular proteins, are known to promote unfolding at acid and alkaline pH values respectively [67,68-j. Here, the unfolding process is at least partially driven by the high dielectric permittivity of the solvent relative to the protein interior.

AC;;, = i’_., - 7% - yw+?rsv= -~y,(l+cos@ (3)

where ysW is the solid/water interfacial tension, y,_, is the surface tension of pure water, AG,, is the reversible work per unit area required to form the solid/water interface, and nsV (= ‘is - r,,) is the surface pressure due to adsorption of water molecules from the vapor (v) phase on the solid. For low energy surfaces (-i, < 50mN m-l), which in&de most polymeric materials, nsV %O. As shown in Table 4 for a series of polymers, all three of these parameters provide a sensitive gauge of surface hydrophobicity.

4. ~hara~teri2ation of the system before adsorption

For (low energy) surfaces where no,~Oo, the polarity of the sorbent surface can be resolved further by assuming that the dispersive (d) and polar (p) contributions to AG,, are additive:

4.1. The adsorbent/aqueous-solution interface

The characteristics of the adsorbent/aqueous- solution interface most relevant to protein adsorption are its specific surface area, hydrophobi~ity and electrical state.

Specific surface areas of sorbents can be determined by a variety of methods, including BET isotherms, dye-binding studies, photon-correlation light scattering and electron microscopy [69]. In general, these various techniques will yield similar, but not identical surface areas for a given sorbent. These unavoidable differences are a reflection of the inherent polydispersity and surface hetero- geneity of the sorbent, as well as artifacts of the experiment itself. For instance, crevices in the sorbent which are accessible to nitrogen molecules (in a BET experiment) may not be visible in an electron micrograph of the surface.

AG,, = AC& + AGiW (4)

Such a division is arbitrary and, since thermodynamics is concerned only with the total work of hydration, AG,,, additional (non-thermodynamic) information usually in the form of an approximate “molecular” theory is required to estimate AGiW and AG&. For instance, Fowkes’ approximation is often used to describe the dispersive contribution

A number of parameters have been used to

Fig. 8. Balance of interfacial tensions (7,,, ylr and y,J acting on a smile drop of liquid (I) on solid (s) with contact angle 8: Young’s Law.

Table -I

Comparison of various scales for characterizmg the hydrophobtcrty of solid (sorbent) surfaces m aqueous solution

Surface

Glass” Mica” Poly(methyl methacrylate) (PMMAY

Polystyreneb

Polyethyieneb

Polypropyleneb

Fluoroethylene polypropylenea

TeRonb

0, {advancing) ;; A&w (deg) (mJ m-‘1 (mJ m-‘)

<5 135.0 _

<5 135.0 12 40.6 - 94.5 91 42.0 -71.1

105 33.0 - 53.6 110 25.7 -47.3 106 21.0 - 52.7 117 18.0 -396

“Data taken from Ref 70.

bData taken from Ref. 71.

[ 16, 721:

AG:, = -2&$$ (5)

where subscript 1 refers to the pure Iiquid phase, and the geometric-mean combining rule originates from the xc-z (where .Y is distance) decay of dispersion forces between semi-infinite parallel fat surfaces. Incorporation of Fowkes’ theory into Young’s Law (Fig. 8) yields

JZ 1 AGii cos $=I J’+--

which, for purely apolar liquids where G,4 = 0 and ‘iI = $, reduces to

(COS @apo~at liquid = 28 - - 1

Application of Eq. (6b) to contact-angle data for an apolar liquid (with known $) on solid (s) yields 7:. and thus AC&.. Apolar Iiquids which are frequently used for such measurements [71] include diiodomethane (2: z y1 = 50.8 mJ m-‘) and ~1- bromonaphthalene (yd zz ̂ /, = 44.4 mJ m-?). Once 7: is known. AGrW cak be determined using Eq. (6a) and contact-angie data for water (;1,= 72.X mJmwt, yp= 21.8 mJ m-l) on the solid at 25’C. The ratio AG,“,,AGtW is often used as an indicator of the polarity of the sorbent surface.

Essentially all interfaces in aqueous solution carry an electrica charge. The charge may origi-

nate from ( 1) association or dissociation of covalently bound surface groups, and/or (2) specific adsorption of low molecular weight ions from the aqueous solution. Type ( 1) surfaces include most biotogical materials, where ionizable surface groups include carboxyl, amino, imidazole and phosphate. all of which associate with protons. Synthetic materials can be of type ( 1) and/or type (2), depending on the raw materials and the method of preparation.

Specific adsorption of ions implies that the non- electrostatic forces driving adsorption are of sufficient strength to allow ions to overcome and create a net electrostatic potential. Ionic surfactants are a well-known class of specifically adsorbing ions; as a rule. small fractions of low molecular weight ions also adsorb specifically to solid/water interfaces.

Under equilibrium conditions, electroneutrality requires that the charge on the sorbent surface be balanced by the net charge of the solution adjacent to it. Thus the sorbent/solution interface is the seat of an electrical double layer. The most generally applicable model for the electrical double layer was derived by Gouy [73,74] and later modified by Stern [75]. In this model (see Fig. 9). the sorbent;‘solution boundary is set at a hypothetical surface, .K = 0, containing all sorbent surface charge. In a system containing no specifically adsorbed ions. there is an ion-free layer adjacent to the

Fig. 9. Schematic representation or the Gouy-Stern model of an electrica double layer. See text for explanation of symbols.

sorbent surface which extends to x = d, where d is the distance of closest approach of a hydrated ion to the sorbent surface. In the case of specific adsorption, the sorbent charge at .u=O is compensated by specifically bound counterions located at x = m and a diffuse charge located at .Y 2 d so that

(ro= -((o,+-LTd) (7)

and hence (r, = - (ge f cid), where cue and od are the surface charge densities at planes x =O and x = d respectively.

The potential 4(x) across the double layer is related to the surface charge density G(X) by Gauss’s Law. which for flat surfaces is

where eeO is the dielectric permittivity. Thus (P(X) can be calculated from knowledge of G(X). Within the charge-free regions 0 <.u -cm and m-c .Y cd, #(x) drops Iinearly with I and its value at any .X within those regions can be calculated from oo, Gd and the theory for a plate condenser. Typically, rro is determined by potentiometric or conductometric titration of the surface using a convenient reference, such as the point of zero charge (i.e. the point at which covalently fixed charges on the sorbent surface .K = 0 balance exactly) of the surface, to fix its numerical value. Determination of Go iS k!SS

precise; it is usually assumed to be the negative of

the electrokinetic charge density G,~ at the hydrodynamic slipping plane (i.e.. ~~ + CT, = - cd 2 CT,&

The validity of this approximation is mostly based on experimental evidence that the zeta potential i, which is the potential at the hydrodynamic slipping plane, deviates substantially from $+, but resembles (#J+ Both i and b& can be determined from electrokinetic measurements [ 161.

For the region s 2 d, Gouy assumed that counterions to the charged surface are exponentially distributed. This led to the well-known diffuse- double-layer model which, for not too large values of $d (Q 50 mV), is given by

&.u) = 4,l exp[ - h.(.x - cf)] (9)

where K is the reciprocal Debye length. Discontinuities in #(.Y) at x = m and x = d arise if the dielectric permittivities of the adjacent regions are not identical. In the Stern layer (0 <.u < d), water ordering at the sorbent surface can substantially decrease the dielectric permittivity of the region. Typically, cStern = 5 - 10, whereas E= 78.5 for bulk water at 25°C.

4.2. Prorcin mo~ecl~les in apeous solution

The complex internal and surface architectures of globular proteins make it difficult to pinpoint those characteristics of proteins in aqueous solution which are of primary importance in their adsorption behavior. Surface properties are clearly important since portions of the protein exterior will initially have the closest and strongest interaction with the sorbent. Important protein surface properties include the effective surface area (size). the surface hydrophobicity and charge distribution, and the presence and number of any surface groups which can specifically associate with groups in the sorbent/solution interface.

Molecuiar graphics calculations on proteins of known crystal structure can provide fairly accurate estimates of surface area. A crude estimate of surface area can also be obtained from a protein’s effective ellipsoidal dimensions. In the literature on protein adsorption, however, molecular weight and

log,,(MW) remain the most common indicators of protein size, possibly because these data are more readiiy available (see, for example, Andrade et af. [X5]); this crude gauge of protein size is only reliable if the proteins under investigation have comparable shapes and specific densities.

Effective surface hydrophobicities of gtobular proteins can be estimated by a variety of techniques, none of which has received universal acceptance. For a protein with known crystal structure. Eisenberg’s atomic solvation parameters (see Fig. 3(c)) provide a fairly reliable estimate of surface (and globaI) hydrophobic~ty [37,77]. Other methods for estimating surface hydrophobiciiies include 0 - ion potential calculations over the accessible surface area of the protein [38], interfacial tension measurements [ 783, hydrophobic- interaction chromatography [ 79,80], and protein partitioning in aqueous-organic two-phase systems [gl f. These approaches, however, are based on a protein’s interaction with either an aqueous/solid or an aqueous/organic interface, on which the protein is likely to adopt a conformation different from that of its native state in aqueous solution; results must therefore be interpreted with caution

since conformational changes in a protein may lead to exposure of its largely hydrophobic interior. Fluorescent dyes which selectively bind with exposed hydrophobic residues are also used as indicators of surface hydrophobicity [SZ]. For two of the most commonly used dyes, nile red and cis- parinaric acid, there is some evidence that binding

induces local, but not global changes in protein structure [83-8Sf. Despite these possible sources of error, surface-hydrophobicity scales for globular proteins are largely in agreement (see Table 5).

Molecular graphics calculations (e.g. Fig. 3(b)) provide a powerful technique for visualizing surface-charge distributions on proteins. The presence of surface-charge asymmetry can also be deduced from measured or calculated dipole moments, but the availability of such data is limited [SS].

Many global properties of proteins are also thought to infhence their adsorption behavior. Of primary importance are the stability of the native state, the relative amounts of ordered secondary structure (r-helix and P-sheet), the overall hydrophobicity, and the electrical state of the protein under the system conditions.

Folded-state stabilities of globular proteins are

Table 5 Comparison of various scales for characterizing hydrophobicities of (hydrated) globular protein surfaces in aqueous solution (data taken or calculated from Refs. 38 and 79-82)

Protein Dimensions (A-“)

Fraction of surface not accessible to o- of K-l&Y

r, (mm)

cis-PnX bindined

Superoxide dismutase 72X40X38 K51 Cyiochrome c 25X25X37 OSi 0.6 ~~yogIobln 44x44x25 0.52 0.8 R~bonuclease 38X18X22 0.54 1.6 Conalbumin 6.3 Ovalbumin 6.5 Lysozyme 45X30X30 0.59 8.5 /?-Giucosidase 15.6 r-Chymotrypsin 16.6 Bovine serum albumin 116X27X27 19.5

- 11.2 Il.1 31.3 -

29.5 69,6 _ 113 59.6 206 49.6 251

_ s 1.3 -

68A 1420

“Ref. 38. bRef. 80. ‘Ref. 79. dRefs. St and 53.

CA. Haynes and Ct: NordelColloids Surfaces B. Btointerfaces 3 (1994) 517-566 531

usually reported in terms of their molar Gibbs

energies of denaturation AN_,,G (kJ per mole protein). However, molecular weights of globular proteins vary widely, making the specific Gibbs energy

of denaturation (kJ g-‘) a more reliable indicator

of relative stability. Other often used scales of native-state stabilities include the temperature Tb (“C) and the added denaturant concentration [den],,, (M) at which 50% of the protein molecules in a sample are denatured.

The percentage of ordered secondary structure in a protein gives some indication of the amount

of conformational entropy the protein gains by (partially) unfolding at the solid/liquid interface. Transmission circular dichroism and crystal struc-

tures from X-ray diffraction studies are the most

common routes for determining cc-helix and /J- sheet contents in native states.

Surfaces of globular proteins are heterogeneous, flexible and highly irregular. Thus some aspects involved in characterizing the electrical state of a

smooth and rigid sorbent surface cannot be applied to the protein/solution interface. For instance, the

assumption that cd% - bek, which allowed us to

apply the Gouy-Stern model to the sorbent/solution double layer, is clearly not valid

at the irregular protein/solution interface.

However, the concepts of proton charge and electrokinetic charge, which is the total charge within the hydrodynamic slipping layer, are valid and their determination as a function of pH provides a reasonable characterization of the electrical state

of a protein. Both the proton charge and the electrokinetic charge of a protein gives some indica-

tion of the electrical contribution to the stability of the folded state (see Section 3.5). In addition, the electrokinetic charge or, more specifically, the electrokinetic potential determines the sign and

strength of electrostatic interactions between the protein and other charged components in the system. The ability of most globular proteins to adsorb ions specifically caii lead to substantial differences in their proton and electrokinetic

charges; this is particularly true for ion-transport

proteins such as human serum albumin (see Fig. 10).

4.3. The aqueous medium

Hydrogen-bond, dipolar and quadrapolar interactions govern the structure and hence the physical properties of water. Liquid water is characterized by a high dielectric constant which effectively screens electrostatic forces, and a relatively high boiling point which indicates that intermolecular forces between water molecules are extremely strong. The relatively open, ordered structure of liquid water is easily altered by the presence of foreign molecules despite the strong intermolecular forces holding it together. As discussed in Section 3.1, excess ordering of water molecules around apolar solutes is largely responsible for the unusually low solubility of hydrocarbons in water at room temperature. Small, highly charged ions also promote local ordering of water; such ions are soluble in water because of highly favorable ion-dipole interactions. In contrast, large monovalent ions exert a disordering influence on water structure [86]. These unique properties suggest

Fig. 10. pH dependence of proton charge (Z,‘) and electroki-

netic charge (Z,,) of human serum albumin dissolved in

50 mM KNO,.

that water and local changes in water structure

(which affect local dielectric properties) play an

important role in protein adsorption.

5. The question of adsorption reversibility

Here, we are referring to the question of whether

the adsorption of proteins on solid surfaces is an

equilibrium or a non-equilibrium process with

respect to sorbate dilution (or addition) under

otherwise constant conditions. In other words, are

protein molecules in the bulk solution free to

exchange with protein molecules of rhe snrne kind

(i.e. the same structure and chemistry) on the

sorbent surface’? The answer, which is all too often

ignored in the literature, determines what thermo-

dynamic criteria apply to the protein adsorption

process and also provides some indication of the

affinity of proteins for solid/water interfaces. In an

equilibrium adsorption process, dilution of sorbate

in the bulk phase creates a transient difference in

the chemical potential of the sorbate at the inter-

face and in the solution. This chemical potential

difference Aus is then eliminated (and equilibrium

is reestablished) by spontaneous desorption of

sorbate. Although such adsorption processes may

not be strictly reversible (since no spontaneous

process is completely reversible), their equilibrium

properties can nevertheless be described by the

laws of reversible thermodynamics [ 871.

In non-equilibrium adsorption, dilution pro-

motes little or no desorption even though the

driving force for desorption. AuS, is non-zero; the

system reaches a metastable state where the energy

barrier to further desorption is prohibitively high.

Thermodynamic description of non-equilibrium

adsorption processes must be based on the laws of

irreversible thermodynamics.

3.1. Adsorptiorl isotherm

The most common presentation of adsorption

data is the adsorption isotherm, where, at constant

temperature. the amount of sorbate adsorbed (T)

is plotted against the steady state concentration c,

of sorbate in the bulk solution after adsorption.

Fully measured adsorption isotherms provide a

convenient method for determining whether an

adsorption process can be treated as reversible

[88]. For reversible adsorption, the ascending

(increasing sorbate concentration in the bulk) and

descending (decreasing sorbate concentration)

branches of the isotherm must overlap at all values

of c,. Reversibility is commonly observed in

adsorption of small molecules. such as monovalent

ions, on solid surfaces. For example, Fig. 1 l(a)

shows an adsorption isotherm for stearic acid (in

carbon tetrachloride) on Graphon at 25’C where

the two branches of the isotherm are indistinguish-

able [89]. Here, adsorption of the sorbate is likely

to involve a single sorbate-sorbent contact. The

strength of this contact, often called the adsorption

exchange (Gibbs) energy xS, is determined by the

difference in sorbate-sorbent and solvent-sorbent

interaction energies [91].

In contrast, adsorption of (uncharged) random-

coil polymers on solids is rarely reversible. For

-i 90 m

e 2 60

0 3 6 9 c lg dm-’

0 2 4 6 6 0 01 02 0.3

Fig. 11. Ascendmg and descending adsorption isotherms for (a)

stearic acid from carbon tetrachloride on Graphon at 25-C

(from Ref. 89. with permtssion). (b) t%o molecular wetght

fractrons of rubber from n-heptane on carbon black at 25’C,

(c) [3H]-phosphorylase b, on butyl Sepharose at 3’C (from

Ref. 90. repnnted utth permtssion), and (d) bovine serum albu-

min on glass (borosthcate) powder dispersed in 0.05 hf phos-

phate buffer at pH 7 and 25.C (from Ref SSI.

example Fig. 11 (b) shows adsorption isotherms for two molecular weights of rubber from solution in n-heptane onto carbon black at 25 ‘C where a hysteresis is observed over a wide range of c, L-923. Because of their great size and flexibility. polymers can make numerous contacts with a sorbent surface upon adsorption [93]. The number of contacts made is governed by the magnitude of xs, which in this case refers to the exchange energy characterizing the interaction of a polymer segment with the sorbent, and the conformational entropy loss associated with constraining the rotational freedom of the polymer upon adsorption. Thus not all polymer segments form contacts with the sorbent surface (unless xs is extremely large); instead, many segments are located in flexible loops and tails which extend into the bulk solution. Even so. the number of contacts is usually substantial. For instance. the formation of 50 segment-sorb~nt contacts would involve only 5% of the segments in a lOOO-segment polymer chain. Thus, even if the effective contribution (including entropy losses) from each of these contacts to AadsG is relatively small, say - 2 kJ per mole contact, the total driving force for adsorption will be Iarge (e.g.- 100 kJ per mole polymer).

If ;cs is small. dilution could promote detachment of a few of the polymer segments contacting the surface (Ref. 91, Chapter 5). Each such segment detachment will lead to an increase in the conformational entropy of the adsorbed polymer, which in turn decreases the driving force for further segment desorption. A substantial driving force is therefore required for simultaneous desorption of all bound segments on a given chain. On its own, dilution rarely provides such a driving force. Instead, displacer molecules. such as heparin or hydrophobic polymers, which effectively compete for adsorption sites on the sorbent surface are usually needed to desorb non-ionic polymers from solid/liquid interfaces (Ref. 91, Chapter 4).

Proteins are polymers. They too can form numerous contacts with a sorbent surface. However, globular proteins are not random coils. As discussed in Section 2. native states of globular

proteins in aqueous solution are highly ordered: most of the polypeptide backbone has little or no rotational freedom. Thus formation of the first few protein-segment-sorbent contacts may induce an increased rotational freedom (i.e., conformational entropy) in other parts of the polypeptide backbone. For instance, contact formation could coincide with the partial breakdown of an z-helix or a P-sheet, which would then promote further contact formation until the flexibility of the adsorbed protein reaches an optimum. After compiling data from a variety of sources and experimenta techniques, Norde [ 161 concluded that layer thicknesses of proteins adsorbed to solids are usually comparable to the dimensions of the native protein in aqueous solution. This suggests that although structural rearrangements may occur upon adsorption, the internal coherence (i.e. stability) of globular proteins prevents them from completely unfolding on a surface into loose “loop and tail”- like structures. Thus the number of protein- segment-sorbent contacts formed at steady state is determined by a subtle balance between intermolecular and intramolecular forces. For instance. adsorption of a highly stable globular protein (e.g. lysozyme) on a surface to which it is only mildly attracted (e.g. hydrophilic surfaces such as silica) would probably involve a relatively small number of segment-sorbent contacts.

Although data are extremely limited, there is some experimental evidence that adsorbed proteins make large numbers of contacts with solid sorbents. For various blood proteins on silica, Morrissey and Stromberg [94] used IR spectroscopy to estimate the number of backbone- carbonyl-sorbent contacts formed at steady state. At pH 7.4, about 11% of the >C=O groups in albumin were in contact with the silica surface; at pH c 6, the percentage of bound carbonyl groups increased to 18%. For fibrinogen, as much as 20% of the backbone carbonyl groups were in contact with the sorbent surface.

For uncharged random-coil polymers, multiple contact formation leads to irreversible adsorption. The same is true for proteins. Figures 1 l(c) and

53-t CA. Hclynes and K I;orde Collolds Sttrjuces B: Bloznrerfuces 2 ( 1994) 31 i-566

11 (d) are adsorption isotherms for [3H]-phosphorylase b, on butyl Sepharose at pH 7 and 25 ‘C and human serum albumin on glass at pH 7 and 25’C respectively. For both systems, adsorption is a non-equilibrium irreversible process, as is the case for most (well-studied) protein adsorption systems [ SS,90].

5.2. The ~rre~ersi~~e entropy ~rodl~ction An&Sir upon adsorption

In general, thermodynamic description of protein adsorption processes should be based on the laws of irreversible the~odynamics. Regrettably, much of the previous work in this area is erron- eously based on reversible thermodynamics. The most common example of this dubious practice is the determination of Aad,G by fitting (ascending) protein sorption data to the Langmuir isotherm equation (see for example Refs. 9597), which is only applicable to reversible adsorption. Another common approach is that proposed by van Oss [71,98], which involves the determination of the interfacial Gibbs energy of interaction AsPwG through the DuprC equation:

AspwG = ‘isP - ‘isw - ‘/pw (10)

which represents the reversible work of forming a sorbent (Q/protein (p) interface in aqueous (w) solution at constant temperature and pressure. In this case, it is not clear what relation Asp,,,G has to AadsG since the latter energy change describes an irreversible process which involves structural per- turbations in the adsorbed protein. In Eq. (lo), the sor~nt/protein interfacial tension ysp is determined from contact-angle data and application of Young’s law (Eq. (3)), which also is based on equilibrium (reversible) thermodynamics and the implicit assumption that the chemical and structural states of the protein molecules on the sorbent surface are the same as in solution.

The minimum errors associated with treating protein adsorption as a reversible process can be estimated by calculating the irreversible contribution to the adsorption entropy A_&$,. In a closed

system, the entropy change associated with any internal process, reversible or irreversible, can be written as

dS=~+dSi, (lla)

where for a reversible change d&,=0, and for an irreversible change dS,>O and QreY is the reversible heat exchange between system and surroundings and Q,,,IT = dS,,,. Following Everett [99], we can calculate Aad& from the area of the hysteresis loop in an adsorption isotherm:

Ldir = R Tpo

r* d In c, (1lf-v

where r* is the adsorbed amount at the upper closure point of the hysteresis loop. Accurate solution of this integral requires detailed knowledge of the ascending and descending branches of the isotherm in the dilute c, region. Unfortunately, such data are rarely available. However, a minimum value for dadsSir can be established by reset- ting the lower closure point of the hysteresis loop at the lowest detectable limit for c,. Using this approach, Jennissen [90] found that A,d,S,,>42 J mol-’ K-’ for the isotherm shown in Fig. 11 (c).

An entropy gain of 42 J mol-’ K-i corresponds to an irreversible Gibbs energy change (AadsGu= - TA,,,Si,) of- 12 kJ mol- ’ at 300 IS. This loner limit for AadsGlr is non-negligible when compared with most published AspwG data [71], indicating that irreversible entropy (and enthalpy) changes cannot be ignored in thermodynamic descriptions of protein adsorption.

6. Characterization of adsorption isotherms for

model systems

6.1. ~escripf~o~ of model systems

Investigations on simple “model” systems, consisting of a well-characterized protein, a well- characterized sorbent, and an aqueous solvent

CA. Haynes and R’. R;ordejCollords Surfaces B. BlomreTfnces 2 ( 1993) 517-366 535

containing only simple ions, have provided the most reliable and meaningful data on the thermodynamics of protein adsorption [loo]. In most cases, the system is unbuffered so that pH changes and proton transfer reactions can be monitored as a function of adsorbed amounts. Results from such studies are the basis for most of the general principles which are thought to govern the protein adsorption process; unfortunately, even for these simple systems, a complete picture has not yet emerged. Nevertheless, resolution of the interactions governing adsorption in these model systems will provide the only reliable foundation for understanding protein adsorption in more complex, technologically important processes.

For protein adsorption at solid/liquid interfaces, only five model-system studies [26,27,101,102) are of sufficient detail to provide an understanding of the thermodynamic driving force for adsorption. Four of the studies involve small single-domain globular proteins: hen egg-white lysozyme (LSZ), bovine pancreas ribonuclease (RNasef, myoglobin (MGB), and calcium-containing cr-lactalbumin (rLA) from bovine milk. These four proteins are of similar size, shape and specific density (approxi-

mately 1.4 g cme3), but differ in surface properties, total hydrophobicities and native-state stabilities (see Table 6). On the Eisenberg scale, the total hydrophobicities of the proteins increase in the order RNase < LSZ < crLA c MGB; based on dye binding studies, their surface hydrophobicities increase in the order MGB < aLA < RNase < LSZ [82,84,85]: AN-DG data from micro-DSC studies indicate that their native-state stabilities increase in the order crLAc MGB <RNase<LSZ. The isoelectric points for the four proteins are also different, which indicates that their net electrostatic interaction with a sorbent surface will differ at a given pH and as a function of pH.

The fifth modei-system study involves human serum albumin (HSA), a multi domain blood protein of comparatively large size and molecular weight (see Table 6). HSA has a relatively low native-state stability as inferred from its large conformational adaptability to changes in environment [ 1033. Its large size and low surface- area/volume ratio suggests that its surface is relatively hydrophilic, although its tendency to dimer- ize and to adsorb low molecular weight polymers and fatty acids makes it difficult to characterize

Table 6 Some physic~hemical properties of the five model proteins: hen’s egg-white lysozyme (LSZ), bovme pancreas ribonuclease (RNaset, sperm whale myoglobin (MGB), z-lactalbumin from bovine milk (zLa), and human serum albumin (HSA)

Property LSZ RNase MGll zLA HSA

Molar mass (D) Dimensions (nm3) Diffusion coefficient (m’s_‘) Isoelectric point Total hydrophobicit~ (J g-l) % protein surface which is apolarb

AK-DC (J 9-l) Thermal’ Denaturant” Secondary structure

% x-helix % /?-sheet

14600 13680 17800 14200 69000 4.6 x 3.0 x 3.0 3.8 x 2.8 X 2.2 4.5 x 3.5 x 2.5 3.7 x 3.2 x 3.5 12 x 2.7 x 2.7 1.04 X 10-10 1.26 x 1O-1o 1.10 x lo-‘0 1.06 x lo- lo 0.70 x lo-I0 11.1 9.4 7.0 4.3 4.6 -7.6 -8.7 -4.1 -5.8 -3.8 59 54 52 -

+4.1 + 3.2 + 2.g + 1.5 +4.0 t3.9 +3.1 +1.9

42 11.5 75 26 70 33 14

A.GD refers to the change due to the transition N-D. a Ref. 37. ‘Ref. 38. c Ref. 52.

the surface polarity accurately [ 1041; it has the largest total hydrophobicity of the five model proteins.

Sorbents used in these model studies include positively charged and negatively charged polystyrene (PS) latexes, uncharged polyoxymethylene (POM), silver iodide sols (AgI), gIass and hematite (r-FezO,), which can carry a positive or a negative charge depending on pH (see Table 7). The relatively low < potential for the positively charged PS surface may be due to the specific adsorption of PO:- and HPOi- ions within the hydrodynamic slipping layer. The hydrophilic nature of the z- Fe20, surface was established using the water- vapor sorption data reported by McCafferty and Zettlemoyer [ 1051.

6.2. Compnrisorr ofadsorption isotherms

Although they should not be used to determine AadsG, ascending branches of adsorption isotherms

Table 7 Some physicochemica1 properties of various solid sorbent surfaces

can provide information on the relative affinities of proteins for a given interface. For instance, Fig. 12(a) shows (ascending branches of) adsorption isotherms for small model proteins on positively charged PS in 50 mM phosphate buffer at pH 7.0 and 25 ‘C. The initial slope of each isotherm is near infinite, indicating that all of the proteins have a high affinity for the interface. High affinity

isotherms are typical for systems where the sorbate can easily form multiple contacts with the sorbent upon adsorption [ 1061. Consequently, random- coil polymers usually show high affinities for (solid) interfaces. For proteins, descending branches of the adsorption isotherm are almost always of the high affinity type, but gentle initial slopes are sometimes observed in the ascending branch of the isotherm (e.g. Fig. 11 (c)). Unfortunately, no consistent molecular picture has been put forward which explains why proteins with low affinities for a surface (in the ascending branch) show very high affinities for the surface upon dilution. The popular

Disperstons 0.05 M electrolyte

Phosphate buffer Acetate buffer Borate buffer pH 7.0 pH 5.5 pH 9.5

Ps- es+ POM Glass r-Fe,O; r-Fe20;

Nature of charged groups -0so; =+NH- - -o- -OH; -0- Surface charge density (mC m-*) -73 +27 Uncharged ? ? ?

Electrophoretic mobtlity (10s m2 V-” s-‘) --tY +2.3 -0.5 -3.9 + 1.3 -2.9 Electrokinetic potential (mV) -69 +3:! -6 -51 f20 -47 Hydrophobicity (contact angle of a se&e drop

of 0.05 M phosphate buffer) 8’- 52; 6-t. 0’ HydrophilIca Specific surface area (m’ g-‘f 10.0 12.4 30.0 0.5 36.0 36.0

Macroscopic surfaces 0 01 M electrolyte; phosphate buffer pH 7.0

Glass SiO, PSgiass PSSiOl

Thickness of the PS layer fnm) Streaming potential (mV) Electrokinettc potential (mV) Hydrophobtcity (contact angle of a sesstle

drop of 0.01 M phosphate buffer)

? _ 35 -4.5 -3.9 - I.9 - 1.9 -18 -21

Hydrophilic 84; 52;

“Mcfafferty and Zettlemoyer [ 105].

C.d. Haynes and W’. .Vorde’Colloids Surfaces B. Btomterfhces 2 (1994) 317-566

2 pH70 PS. ~.+32mV pH7.0 PS- Is-69mV

I I I I I I I L I I I I I I 0 02 0.4 0.6 0.5

0. 0 0.1 Q2 a3 OL 05 06

c,lgdm-’ cslg dm-’

Fig. 12. Adsorption isotherms for (a) Iysozyme (0). ribonuclease (0). myoglobin (x), and z-lactabumin (0) on positively charged

polystyrene in 50 mM phosphate buffer at pH 7 and 2S’C, and for (b) human serum albumin (A), lysozyme (0). ribonuclease (‘2).

myoglobin (x) and z-lactabumin (0) on negatively charged polystyrene in 50 mM phosphate buffer at pH 7 and X’C. Also shown is the relative proton charge on each protein at pH 7. Data from Haynes and Norde [26]. Arai and Norde [27] and Norde [IO?].

explanation is that these proteins initially form a relatively small number of contacts with the sorbent surface, but after adsorption they slowly undergo structural alterations which enhance binding significantly. However, beyond an adsorption induction period which can last up to 3 h. ascending branches of adsorption isotherms are usually independent of equilibration time, even in the dilute c, region. An alternative explanation is that these proteins form molecular clusters on the solid surface after adsorption; thus desorption involves the detachment of a cluster of proteins rather than desorption of a single protein molecule. There is limited data supporting this picture. For instance, from transmission electron microscopy images, Schakenraad et al. [ 1073 concluded that fibronectin forms molecular clusters upon adsorption to Teflon. However, Teflon is very hydrophobic; consequently, native fibronectin has a very high affinity for bare Teflon.

In theory, maximum surface concentrations for unperturbed globular proteins adsorbed in a side- on orientation range from about 1.5 to 8 mg m-’ [108-l 111 depending on the precise size and shape of the molecule. In contrast, a maximum surface concentration of 0.55 mg m-* is predicted for a fully adsorbed (i.e. all segments form contacts) polypeptide chain where the side-on surface area of each residue is 0.3 nm’. As shown in Fig. 12(a), protein adsorption isotherms tend to reach well-

defined plateaux at high c,. Measured plateau values for small globular proteins are almost always less than 2.5 mg rne2, suggesting that (1) some proteins form incomplete monolayers on solid sorbents and/or that (2) some proteins (partially) unfold and spread on sorbent surfaces upon adsorption.

Figure 12(b) shows ascending adsorption isotherms for several proteins on negatively charged PS in 50 mM buffer at pH 7.0 and 25 ‘C. Again. all of the isotherms reach well-defined plateaux at high c,. However, a step or “kink” is evident at an intermediate c, in the isotherms for xLA and HSA. Such “kinks” are not uncommon in protein adsorption and, when present, indicate that protein binding to the surface is bimodal. A reasonable, although largely untested explanation of this phe- nomenon was forwarded by Fair and Jamieson [ 1121 when investigating the adsorption of blood proteins on polystyrene latexes. They propose that protein molecules adsorb in a random, independent manner until the surface concentration of protein reaches a critical value (characterized by the high- affinity lower plateau). At high cs, intermolecular nucleation at the sorbent surface causes the formation of a two-dimensional protein crystal, thereby increasing I. The intermediate “kink” region therefore corresponds to a two-dimensional disorder- order phase transition in the protein on the sorbent surface. Unfortunately, experiment has not yet

established the validity of this model. For Instance.

the model predicts that the tendency for proteins

to desorb will decrease once they have reached the

glassy state (i.e. the second plateau) on the surface.

Such behavior has indeed been reported [ 1011,

but the reverse behavior has also been observed

[110.113].

An alternative explanation for the plateau-value

kink is that the orientation of the adsorbed protein

varies with surface coverage. A side-on to end-on

orientation transition could explain the observed

kinks in Fig. 12(b) [ 1141. Evidence for an orienta-

tion transition has come from surface force meas-

urements on albumin on various hydrophilic

surfaces [ 115.1161. The orientation of albumin is

side-on at low salt concentrations whereas a sig-

nificant fraction of the molecules adsorb end-on in

0.15 M NaCl. The same group also used the surface

force technique to explain kinks in adsorption

isotherms for insulin on hydrophilic surfaces by

monitoring the formation of adsorbed multilayers

as a function of insulin concentration in the bulk.

In contrast, albumin adsorbs as a monolayer at all

bulk concentrations.

6.3. Drfluence oj‘sorbent and protein charge

Protein adsorption at a charged surface involves

overlap of the electrical double layers at the sol-

vated surface and the solvated protein surface. This

overlap will result in electrostatic attraction if the

protein macro-ion and the sorbent have opposite

charge sign or in repulsion if their electrokinetic

charges are of the same sign.

Comparison of Figs. 13(a) and 12(b) reveals the

importance of global electrostatic forces in protein

adsorption. On positively charged PS at pH 7

(Fig. l?(a)), adsorption plateau (IPI) values

increase in the order LSZ < RNase < MGB < xLA;

here. the proteins adsorb in accordance with their

net electrostatic attractions (or repulsions) for the

surface. For instance. zLA, which is the only pro-

tein having a net negative charge at pH 7, gives

the largest adsorbed amounts on the positively

charged sorbent. On negatively charged PS (under

otherwise constant conditions), the relative posi-

tions of the plateau values are nearly reversed.

LSZ now has the largest Tp’. which is m line with

its strong global electrostatic attraction for the

surface. However, ILA also has a relatively large

I-P’ on the negatively charged surface despite being

electrostatically repelled from it. 1Moreover. the

plateau value for RNase is similar on the two

surfaces even though the protein carries a substan-

tial positive charge at pH 7. Thus. although global

electrostatic forces undoubtedly affect adsorption.

they do not dominate it.

A second approach for establishing the influence

of electrostatics on adsorption behavior is to vary

the solution pH while holding the electrokinetic

charge density on the sorbent surface constant.

Here. the range of pH values which can be investi-

gated depends upon the nature of the charged

groups on the sorbent surface. Figure 13 shows

plateau adsorption values for serum albumin on

various surfaces for which the electrokinetic poten-

tial or, for that matter, the electrokinetic charge

density is constant over the pH range studied. If

global electrostatic forces between the protein and

the sorbent surface dominated protein adsorption

behavior, we would expect TP’ to be a monotonic

function of pH. Instead, all of the curves exhibit a

maximum near the isoelectric point (~1) of the

protein,‘sorbent complex. This bell-shaped depen-

dence of TP’ on pH is commonly observed in

Fig. 13 Plateau values for the adsorptlon at 2). C of human

serum albumm on negatively charged polystyrene. posmvei)

charged polystyrene. uncharged polyoxymethqlene, and on

negati\el> charged silver iodide. The background electrolyte IS

10 mM KSO, except for the PO&l system where it IS IO m&f

C.d. Ha,vnes und W’. ~Vordr~Collods Surfaces B. Biomerfaces 2 (fY91) 517-566 539

protein adsorption on solid surfaces [ 94,111,117], which again suggests that global electrostatic interactions between the protein and the surface do not dominate protein adsorption phenomena. One explanation for the complex dependence of I@ on pH is that increased lateral electrostatic repulsions between charged proteins on the surface prevents the formation of close-packed monolayers. Thus the bell-shaped curve results from the competition between protein-protein and protein-surface electrostatic interactions. Limited experimental evidence supporting this explanation was provided by Shastri and Roe [ 1181, who found a linear correlation between TP’( pH) for albumin on POM single crystals and the osmotic second virial coefficient B,,(pH) for the protein in aqueous solution.

However, there is now convincing evidence that pH also affects the level to which native-state proteins undergo structural alterations upon adsorption to solid surfaces. For instance, Norde [ 1021 studied the temperature dependence of the initial slope of the adsorption isotherm for HSA on negatively charged PS (Fig. 14(a)). At pH 4.7, they observed an increase in slope upon increasing temperature from 22°C to 37°C. No change in slope was observed upon changing temperature from 5°C to 22°C which indicates that an additional endothermic event occurred in the adsorption process at 37’C. Since the protein carries no net charge at pH 4.7, Norde concluded that the additional endothermic process at 37’C was due to enhanced breakdown of ordered secondary

_. 1.61 L

0 12 E

08 0.6

pH 4.7

structure within the protein upon adsorption; in other words, albumin denatures on the surface at 37’C but maintains (most of) its native-state structure upon adsorption at 22’C and pH 4.7. In accordance, Tp’ was found to be much lower at 37’C than at 22°C or 5’C. At pH 7 (Fig. 14(b)), where the protein has a negative charge and therefore an electrostatic repulsion to the sorbent, substantial changes in slope occur with each temperature change. Combining this result with plateau adsorption data (see Section 3.5) Norde concluded that at 22’C the protein is more unfolded on the surface at pH 7.0 than at its p1 (which is consistent with the protein’s lower structural stability at the higher pH [ 1031. This suggests that the structural states of adsorbed proteins vary as a function of solution pH. Anticipating the discussion in Section 6.6, we mention here that micro-DSC data for LSZ and aLA adsorbed to negatively charged PS indicate that the structural rearrangements in the protein are minimum when adsorption occurs at a pH equal to the p1 of the protein’sorbent complex.

6.4. Influence of protein hydrophobicit)

All protein surfaces are composed of a mixture of hydrophilic and hydrophobic residues (see Section 2). Since the entropic penalty for hydrating non-polar molecules is high, the aqueous solution will seek ways to minimize contact with hydrophobic groups on a protein’s surface. Protein aggregation into dimers and higher oligomers is

0 002 004 OC6 0.08

c, lg dm-’

Fig. 11. Initial slopes of adsorption isotherms for human serum albumin on negatively charged polystyrene (a,= - 15.5 PC cm-‘) in 50 mhl KNO, as a function of adsorption temperature and pH: x1 5’C; l . 21’C; A., 37’C.

one mechanism for dehydrating apolar patches on

a protein’s surface; adsorption to a non-aqueous

interface provides another potential mechanism.

Both processes involve a substantial increase in

entropy and. since AG-x - TAS, a decrease in

Gibbs energy (see Section 3.1 and Fig. 5).

It is therefore reasonable to assume that protein

surface hydrophobicity dictates, at least partially.

a protein’s adsorption behavior. For protein

adsorption at the air/water interface. Wei [SZ]

observed a direct correlation between adsorption

rate constants and protein surface hydrophobicit-

ies. In some ways, Wei’s results are surprising since

the comparisons were made at a single pH, where

each of the proteins studied carries a unique non-

zero electrokinetic charge; thus no precautions

were taken to ensure that electrostatic repulsions

between proteins were the same in all systems.

Nevertheless, a clear correlation with surface

hydrophobicity was observed, which suggests that

hydrophobic dehydration effects dominate electro-

static forces in protein adsorption at the air/water

interface.

Studies related to hydrophobic interaction chro-

matography (HIC) have provided substantial evi-

dence that protein-surface hydrophobicity also

influences protein adsorption at solid/water inter-

faces. Regnier [23] and Gorbunov et al. [ 1191

provide excellent reviews of these important results.

A further understanding of the influence of pro-

tein surface hydrophobicity on adsorption beha-

vior at solid,‘water interfaces, where electrostatic

forces are known to be significant. can be gained

by comparing adsorption isotherms for similar-

sized proteins at their isoelectric points. Ideally,

this analysis would be based on the initial slopes

of the isotherms since the magnitude of the initial

slope is an unambiguous indicator of a protein’s

affinity for a surface. However, accurate measure-

ment of initial slopes is difficult, particularly for

protein adsorption to hydrophobic surfaces where

initial slopes are almost always near infinite. An

alternative but less conclusive approach is to corre-

late surface hydrophobicities with adsorbed

amounts (i.e. rp’ values). In Fig. 1% plateau adsorp-

‘E t X

i? ” 2 . I . 1

I I I , I I

0 2 6 6 8 IO relative surface hydraphobuty (t,lmn)

Fig. 15. Correlatton of plateau values \\lth protein surface

hydrophoblclty for the adsorptlon of hen egg-white ljsoz>me

t 0). bovme pdncrease rlbonuclease ( A). x-lactalbumm ( x L sperm \\ hale mjoglobm (+ ) and superoxclde dlsmutass (WI on

negatl\elv charged polystyrene (ci,,= - 15.5 PC cm-‘) in 50 rnhl

KCI at 3 C and a pH equal to the pL of each protem.

tion data are plotted for several similar-sized pro-

teins on negatively charged PS as a function of

protein surface hydrophobicity. To minimize elec-

trostatic effects. each protein was adsorbed at the

solution pH corresponding to its p1. As shown in

Fig. 15. Tp’ tends to increase with increasing pro-

tein surface hydrophobicity. suggesting that the

driving force for adsorption is directly related to

the surface hydrophobicity of the protein.

However. the I-P’ for rLA diverges strongly from

this trend, suggesting that some other effect con-

trols its adsorption behavior on PS. rLA has an

unusually low structural stability compared with

the other proteins studied; thus structural

rearrangements in the protein may dominate its

adsorption behavior (see Section 6.6).

Although the applicability of the approach to

protein adsorption is questionable. results from

contact-angle measurements [ 711 also indicate a

strong correlation between protein surface hydro-

phobicities and the driving force for adsorption:

calculated Gibbs energies of adhesion tend to

increase with increasing protein surface hydropho-

bicity regardless of the relative hydrophobicity of

the sorbent.

The overall hydrophobicity of a protein may

also influence its adsorption behavior. For protein

adsorption at the air water interface. Birdi [ 1201

C..4. Haynes and it: .Vordr~Colhis Surfacrs B Biomrerjius 2 ( 1994) jl7-566 551

found a strong correlation between the total hydrophobicity of a protein and its tendency to undergo structural alterations upon adsorption; similar results are reported by Hamaguchi [121], Tornberg [ 1231 and Wei [82] for adsorption at the air/water interface, and by Norde [ 161 and Arai and Norde [27] for adsorption on PS latexes. These results suggest that adsorbed amounts will correlate inversely with overall protein hydrophobicities since denaturation and spreading of proteins on a sorbent surface will block potential adsorption sites for remaining molecules in the bulk phase.

6.5. I$uence of sorbent polarity

Correlations between protein adsorption and sorbent hydrophobicity indicate that proteins show maximum affinity for surfaces of intermediate polarity. For instance, plateau values for proteins adsorbed to solids of varying hydrophobicity usually exhibit a maximum around i’s= 30 mJ m-” [l&71]. Baszkin and Lyman [ 1231 studied adsorption of the bovine blood proteins albumin, y-globulin and fibrinogen on a series of relatively hydrophobic surfaces. The overall hydrophobicities of these proteins are comparable, ranging from 5.7 to 6.3 J g-i on the Eisenberg scale. In terms of AG~~/AG& (see Section 4.1), the polarity of the sorbent surfaces ranged from 0.54 to 0.95. For each protein, I’P’ increased and the level of desorption decreased with increasing sorbent polarity.

Although reliable data are limited, FpL appears to reach a maximum (for most proteins) at intermediate values for AGT,,,/AG&. and then declines rapidly with increasing surface polarity. For instance, Horbett and Hoffman [ 1241 studied the adsorption of a series of globular proteins on the hydrogel poly( hydroxyethyethacrylate), where AG&,/AG$w=2.2; Ip’ was relatively low for all proteins studied. Moreover, upon decreasing the water content of the hydrogel (i.e. the polarity), Askill et al. [ 1251 observed a decrease in the desorbable fraction of adsorbed protein. The rp’ values are also low for globular proteins (at their

PI) adsorbed to hydrophilic surfaces such as glass, silica and z-FeZO, [27,101]. This trend is in line with the amphipolar nature of protein surfaces and may reflect the energetic reward associated with matching two amphipolar, heterogeneous surfaces. However, data supporting this observation are limited, which again emphasizes the need for a more systematic study.

6.4. Injluence of protein structural stability

Since their folded conformations in aqueous solution are only marginally stable under the best of conditions (see Fig. 5), globular proteins can usually be denatured by a modest change in environment. such as an increase in temperature or pressure, a change in pH, or the addition of small amounts of a denaturant (e.g. urea or guanidinium chloride).

Protein stabilities can also be disrupted by the introduction of a foreign surface or interface to the system. Observations of changes in protein conformation at air/water and oil/water interfaces have been reported since the early part of the century. Excellent reviews of this early protein adsorption literature are provided by Chessman and Davies [126] and Cumper and Alexander [127]. More recently, MacRitchie [ 1281, MacRitchie and Alexander, [ 1291 and others [82,122,130,131] have indirectly probed conformational changes in proteins at the air/water interface by measuring steady state and time-dependent surface pressures, tensions and potentials using, for example, the Langmuir trough or Wilhelmy plate technique. Similarly, the drop volume, pendant drop and Langmuir trough techniques have provided convincing evidence that proteins also denature at oil/water interfaces [ 132-1351. Reviews covering these and other aspects of protein adsorption at air/water and oil/water interfaces can be found elsewhere [ 13.1281.

The complex, heterogeneous nature of the solid/water interface has hindered experimental attempts to probe conformations of proteins adsorbed to solids. Moreover, solid substrates often

interfere with spectroscopic signals (e.g. NMR and transmission circular dichroism) from proteins in the adsorbed state. Nevertheless. there is now substantial evidence that proteins undergo structural alterations at the solid/water interface, particularly when the surface is hydrophobic. Vroman was among the first to recognize that proteins denature at solid surfaces [136]; in Blood. he proposed that “those (globular proteins) which can open easily will do so when they see a hydrophobic surface, and will turn themselves inside out to paste themselves with their fatty hearts onto that surface”. Indirect experimental confirmation of Vroman’s hypothesis was provided by Norde [ 1021 in an exhaustive thermodynamic study of the mechanism of human serum albumin and bovine pancreas ribonuclease adsorption on polystyrene surfaces. By comparing adsorption isotherms, dissolved- and adsorbed-state proton titration curves, and enthalpy of adsorption data for the two proteins, Norde concluded that proteins with low native-state stabilities, such as albumin, possess a strong driving force for adsorption related to breakdown of native secondary and tertiary structure; in other words, adsorption is driven by an increase in the conformational entropy of the protein. Similar conclusions have been drawn from (endothermic) shifts in initial slopes of adsorption isotherms as a function of temperature (see Section 6.3) and from reductions in biological activity upon adsorption [137,138].

Direct evidence of protein denaturation at solid/water interfaces is limited [ 139,140]. Norde and Favier [ 1413 used transmission circular dichroism to measure g-helix contents of bovine serum albumin and hen egg-white lysozyme adsorbed on finely dispersed silica particles. A decrease in x-helix content was observed for both proteins upon adsorption (see Table 8). The extent of g-helix breakdown was shown to increase with decreasing native-state stability and with decreasing concentration of protein in the bulk solution (i.e. decreasing surface coverage). This latter trend suggests that the degree of protein unfolding is at least partially determined by the amount of sorbent

Table 8

Percent z-helix content in bovme serum albumm and hen egg-

white lysozymc m aqueous solution and adsorbed to silica

partlcles (values taken from circular dichroism data of Norde

and Favier [ 1431)

Protein Percent r-hehx content

Dissolved

Adsorbed state

Lysozyme

pH 4.0 32

pH 4.7 33

pH 7.0 32

Human serum albumin

pH 4.0 69

pH 4.7 70

pH 7.0 74

r;rpl=O >3 rg-pl= 1.00

1; ‘- 25 rrp’=o.t6 rP= i 00 22 30 .

r/P = 0.2~ r/P= 1.00 28 38

surface area available to the adsorbed protein. Similar results were reported by Kondo et al. [142] for the adsorption of albumin on silica and by McMillan and Walton [143], who found that coagulation factor XII undergoes a structural alter- ation upon adsorption to quartz but fibrinogen does not.

Fluorescence spectroscopy has also provided direct information on the structures of proteins in the adsorbed state [113,144-148-j. For instance, Andrade et al. [ 1491 concluded that the conformation of fibronectin does not change upon adsorption to hydrophilic silica, but changes significantly upon adsorption to chemically modified hydrophobic silica (where the hydrophobicity is determined by the amount of dichlorodimethylsilane covalently bound to the modified surface). Van Wagenen et al. [150] found that albumin and y- globulin undergo significant conformational changes upon adsorption to hydrophilic glass.

Other spectroscopic techniques, such as NMR and electron paramagnetic resonance, have also been used to probe adsorbed-protein structures [ ljl-1531. Unfortunately, the inherent complexity of protein adsorption systems often hinders meaningful interpretation of data from these techniques. Nevertheless. a successful NMR study was reported

CA. Haynes and W’ ~Vorde Colloids Surfaces B. Biomrerfaces 2 (1994) jl7-566 543

by Benko et al. [ 1541, who concluded that the heme group of hemoglobin undergoes a conformational change upon adsorption to PS. Moreover, several successful Fourier transform infrared (FTIR) spectroscopy studies have recently been reported [ 155-1571. For instance, Barbucci et al. [ 1581 used the amide III region of the FTIR spectrum to monitor qualitatively the helix structure of albumin adsorbed to germanium as a function of adsorption time; the deconvoluted spectra revealed that the a-helix content of the protein diminishes slightly upon initial adsorption and then continues to diminish for about 1 h, at which time the protein reaches a new (meta)stable structure.

Micro-DSC experiments have provided a wealth of direct thermodynamic information on the stabilities (and structures) of globular proteins in the dissolved state. For instance, Privalov [52] and Pfeil et al. [159] used micro-DSC to establish that the denaturation of small single-domain proteins is a thermodynamically reversible process characterized by large increases in enthalpy and entropy (see Fig. 5). Recently, Haynes and Norde [26] used micro-DSC to probe conformational changes in lysozyme and r-lactalbumin adsorbed on negatively charged PS and cc-Fe,O, surfaces. The direct thermodynamic observables in the micro-DSC experiment are the enthalpy change (AP_&)ads, the denaturation temperature Td, and the heat capacity

change (AP-DCJ~~~ associated with temperature- induced structural transitions in the protein molecules (see Tables 9-12). A large value of (AP_&)ads is indicative of a structural transition that involves a substantial loss of favorable intramolecular interactions and ordered secondary structure within the adsorbed protein molecule in the P state, where P represents the “perturbed” structure of the native protein on the sorbent surface. A high Td is indicative of a highly stable P-state structure. Comparison of the P-state denaturation data shown in the Tables 9-12 with the corresponding N-state data (not shown) reveals that the P state is always less stable than the N state and that significantly fewer intramolecular bonds are

Table 9 Thermal denaturation data. obtained from micro-DSC experiments, for lysozyme adsorbed to negatively charged PS in 50 mM KCI (data from Haynes and Norde [26])

Sample pH Apparent T., AP-DH AP_DCI (>C) (kJ mol-‘) I kJ K-’ mol-‘)

2.2 54.1 31.0 2.4 2.45 0.0 - 2.8 56.0 32.1 2.0 2.9 62.1 47.8 2.6 3.95 65.4 59.0 3.0 6.45 68.9 63.6 3.6 6.45 69.5 74.8 3.8 6.6 68.4 84.4 3.7 6.9 63.8 170.3 5.5 9.15 66.8 135.3 5.5 9.4 66.2 104.8 5.4 9.4 66.0 56.5 3.5

Table 10 Thermal denaturation data, obtamed from micro-DSC experiments, for lysozyme adsorbed to hematite in 50 mM KC1 (data from Haynes and Norde [26])

Sample pH Apparent r, A&& &-rXp (‘C) (kJ mol-‘) (kJ K-’ mol-‘)

8.3 72.5 451.8 5.8 8.3 72.8 438.6 6.1 8.8 72.8 448.7 - 8.8 71.6 433.0 5.9 8.95 67.0 417.7 5.7 9.05 65.2 399.9 5.5 9.3 63.0 338.8 - 9.3 63.6 347.0 5.8 9.7 60.5 297.6 5.6 9.7 61.1 301.2 5.6

10.5 58.2 269.9 5.4

broken during transiton from the P state to the denatured state. This difference is particularly striking in the negatively charged PS system, where

(A+& )ads values for both proteins are near zero across the entire pH range. Clearly, both proteins lose most (and sometimes all) of their ordered secondary structure upon adsorption to the hydrophobic PS surface. In contrast, (Ap_&Y)ado data for LSZ adsorbed to the hydrophilic r-FezOJ surface are only about 20% lower than AN-a for the native protein at pH 5.3 (see Fig. 5(a)). Thus the amount of native-state secondary structure lost

Table 11 Thermal denaturatton data. obtamed from macro-DSC eupert-

ments. for x-lactalbumin adsorbed to negatwely charged PS m

50 mhl KCI (data from Haynes and Norde [XI])

Sample pH Apparent r, A%DH A&&P fZC) (kJmol_‘) (kJK-‘mol-‘j

5.5 _ 0 _

5.5 56.5 5.6 2.0 6.0 58.2 4.9 14 6.0 _ 0 _ 6.7 0 _ 6.5 59.6 0 _

7.5 64.5 0 1.5

7.75 61.5 102 20 8.7 53.5 13 5 1.4

8.7 56.1 21.3 1.6

Table 1Z Thermal denaturatton data. obtatned from macro-DSC experi-

ments. for r-lactalbumm adsorbed to hemattte m 50 mM KCI

(data from Haynes and Norde [26])

Sample pH Apparent Td A,-& Ar,-,Cp fC) (kJ mol-‘) (kJ K-’ mol-‘)

5.5 _ 0 _ 5.5 _ 0 _ 6.0 0 _

6.1 _ 0 _ 6.5 0 _

6.5 _ 0 _

7.0 43.8 34.0 2.7

7.6 42.7 4O.S ‘5 _. 7.9 443 63.6 2.5

7.9 45.2 57.3 2.3 8.5 41.1 61.1 2.5

upon adsorption is highly dependent on the nature

of the sorbent surface.

The tendency for a protein to undergo structural

changes during adsorption also depends on the

stability of the protein in the native state. For

instance, crLA, which has a relatively low native-

state stability (T,=61.4’C and AN_&=

158 kJ mol-’ at pH 5.3). retains virtually none of

its compact native-state secondary structure upon

adsorption to r-Fe,O, (see Table 12). In contrast,

LSZ. which is a relatively stable globular protein,

retains most of its native-state structure upon

adsorption to x-FelO,.

For both LSZ adsorption systems. (Ap_a)ads

has a strong dependence on pH. Some of this pH

dependence must come from the deleterious effects

of high protein surface charge on the stability of

the native state (see Section 3.5). However. Fig. 16

shows P-state denaturation enthalpies for LSZ

adsorbed on negatively charged PS as a function

of pH. (Ap_DH )a& is a maximum near the pI of the

protein/sorbent complex (PI s 7.9) rather than at

the pI of the dissolved protein (~1: 11.2). Thus

the stability of the P-state structure is greatest

when the charges on the sorbent surface and the

protein surface cancel exactly. This indicates that

( AP-DH )ad. also depends on electrostatic inter-

actions between protein and sorbent.

The TP’ values for LSZ adsorbed to negatively

charged PS are also shown in Fig. 16. Both curves

(I-P’ and (Ap_&)&) are bell shaped and show a

maxima near the pI of the protein, sorbent complex

(complex pI 2 7.9 from electrophoretic mobility

measurements). As discussed in Section 6.2. this

strong correlation suggests that variations in

adsorbed-protein structures are largely responsible

for the complex dependence of rp’ on pH.

Thermal transitions from the P state to the

denatured state are also characterized by increases

in heat capacity (AP_DCp)ads. This indicates that the

denaturation process leads to substantial hydration

of hydrophobic residues which were buried (and

dehydrated) in the P-state structure. From this we

200- -3 5 r

s loo- 4 ,

0 I I I I _1 2 4 6 0 IO 12

Ftg 16. pH dependence of P-state denaturatton enthalptes

(A&f) and plateau values for hen egg-uhite lysozyme

adsorbed to negattvely charged poly-styrene (~a = - 15.5 uC cm-:) in 50 mhl KCI solutton.

can infer that internal water contents of adsorbed proteins are small. However, (Ap_&‘Jads is always smaller than the corresponding AN_&,. This implies that either (a) adsorbed hydrophobic residues may not fully hydrate during denaturation, or that (b) structural rearrangements in the adsorbed protein molecule lead to the hydration of some residues which are buried and shielded from water in the native state.

Complementary results were reported by Steadman et al. [160], who used micro-DSC to establish denaturation trends for seven globular proteins adsorbed to silica and to several chemically modified silica surfaces. Based on r, data, the stabilities of all seven proteins decreased upon adsorption to unmodified silica with the least stable N-state protein showing largest shift in Td, and vice versa. For lysozyme, destabilization of the P-state structure was found to increase with increasing sorbent-surface hydrophobicity.

Structural rearrangements in proteins adsorbed to solid surfaces usually lead to a fairly compact protein layer rather than to a loose “loop and tail” structure. Evidence for the compact nature of adsorbed protein layers has come from ellipsometry data, which give the optical layer thickness, and from light-scattering and viscometry data, which give the hydrodynamic layer thickness [139,161-1671. For instance, Cuypers et al. [168,169-J used ellipsometry to examine layers of albumin and fibrinogen adsorbed on hydrophobic chromium and hydrophilic chromium oxide surfaces (fibrinogen data shown in Fig. 17). Fibrinogen’s size and ellipsoidal shape are characterized by a major axis diameter of 45 nm and a minor axis diameter of 9 nm. On the hydrophilic chromium oxide surface at pH 7, fibrinogen adsorbes in a 13-14 nm layer and reaches a Tp’ value of 4.5 mg m -‘; these data are commensurate with a side-on orientation. On the hydrophobic surface, l-p’ increases to 8.7 mg m-” and the layer thickness decreases to between 7 and 3 nm; since these layer thicknesses are less than the minor axis of the protein, Cuypers et al. concluded that the adsorbed layer is compact and the conformation

Fig. 17. Thickness (-) and refractive index (---) of adsorbed layers of human fibrinogen on hydrophobic chromium (lower curves) and hydrophdic chromium oxide (upper curves) as a function of time: system contains IO mg fibrinogen per dm3 and 10 mM Tris-HCI buffer at pi4 7. Reprinted from Cuypets et al. [ 1651 with permission.

of the protein differs in the adsorbed and dissolved states. In comparison, albumin, which is characterized by a relatively low structural stability, forms a compact. partially denatured layer on both the hydrophobic PS surface and the hydrophilic TV- FeZO, surface.

As discussed in Section 2, structural stabilities of globular proteins are determined by a complex balance of intermolecular and intramolecular interactions, with hydrophobic dehydration and backbone conformational entropy providing the strongest stabilizing and destabiIizing effects respectively. Most (if not all) of these interactions will be altered by the introduction of a foreign surface. For instance, adsorption to a hydrophobic surface provides a mechanism by which a protein can increase its conformational entropy without exposing hydrophobic residues to the aqueous environment; the probability of this process occur- ring in a given adsorption system would appear to depend on the protein’s native state AN_nG since the structural stability of a protein reflects its internal coherence. The number of protein-sorbent contacts formed upon adsorption will also depend

on AX_&. Figure 18 compares Tp’ values for smail globular

proteins (at their PI) adsorbed to negatively charged PS as a function of A,_,G. The value of

0.5. I 20 #) LO 50 60 70 80

AN_oG I k J mol-’

Fig. 18. Correlation of plateau values with native-state Gibbs energies of denaturatlon ASD G for the adsorption of superoxide dismutase (ml, hen egg-white lysozyme (x ), bovine pancreas ribonuclease (A), sperm whale myogIobin (+ f, and Z- lactatbumin (0) on negatively charged polystyrene (Q= - 15.5 PC cm-?) in 50 mM KCI at 3’C and a pH equal to the pI of each protein.

PL is large for the least stable protein (zLA) and falls sharply with increasing AWDG. LSZ, however, diverges from this trend, possibly because of its tendency to aggregate in solution and on a surface [ 1701; the high I-P’ value for LSZ would therefore follow from its relatively high surface hydrophobicity (see Fig. 15). Interpretation of the remaining data in Fig. 18 is more straightforward. The fPL for rLA reflects the large conformational entropy gain resulting from a loss in ordered secondary structure upon adsorption. For the more stable proteins, MGB, RNase and SOD, this entropic gain is increasingly opposed by the concurrent destruction of favorable (i.e. stabilizing) interactions within the protein macromolecule.

7. Electrostatics of protein adsorption

In aqueous soWion, charged sorbent surfaces and protein macroions are surrounded by counterions. A fraction of these counterions may be specifically adsorbed to the protein or to the sorbent surface; the remaining ions are distributed around each charged surface in a diffuse layer. For instance, Norde and Lyklema [ 171 J found that K’ ions specifically adsorb to negatively charged PS in aqueous KNO, solutions. Similarly, albumin is known to specifically adsorb anions (see Fig. IO).

Thus the electrostatic characteristics of the sorbent and the protein are affected by the chemistry and the concentration of additional electrolyte present in the system. Electrostatic properties of proteins are also affected by solution pH. As shown in Fig. 10, charged residues on the surface of HSA titrate over a wide range of pH.

As discussed in Section 6.3, adsorption of a protein to a charged interface results in an overlap of electrical double layers as well as a change in the polarity of the interfacial region. These environ- mental changes can dramatically affect the properties of the protein (e.g. shift the pK, values of residues adjacent to the sorbent surface) and the distribution and surface concentrations of specifically adsorbed ions. As shown by Norde and Lyklema [172], the contribution of these changes in electrostatic environment to the overall protein adsorption process can be estimated by comparing the electrical properties of the charged species before and after adsorption: proton titrations and electrophoretic mobilities of the individual species and the protein/sorbent complex (as a function of pH) form the basis of this analysis.

7.1. Sarbent surfk+e charges in aqmxw solution

In terms of their electrostatic properties, solid sorbents can be categorized as (I ) uncharged suc- faces (e.g. POM ), (2) surfaces where the charge is determined by ions other than protons (e.g. silver iodide sols), (3) surfaces that contain only strongly acidic or basic groups which titrate at extreme pH values (e.g. sulfonated PS), or (4) surfaces with charged groups that protonate at moderate pH values (e.g. oxide surfaces such as SiOz and z- Fe,&). Sorbent surfaces of types (1) and (2)

provide the simplest model systems for elucidating the effects of coulomb interactions on protein adsorption because the charge on the sorbent surface is invariant with pH. Such systems therefore provide an unambiguous means of constructing hydrogen ion titration curves for proteins in the adsorbed state. Type (3) surfaces may also be used provided the pH range of the proteinisorbent

titration does not coincide with the p&(s) of the charged groups on the sorbent surface.

All globular proteins contain a variety of acidic and basic groups. The number of dissociating groups and their apparent dissociation constants can be accurately estimated from a protein’s hydrogen ion titration curve, which gives the net proton charge on the protein as a function of pH [64]. Changes in a protein’s conformation and local ~nvieonment are often reflected in the hydrogen ion titration curve for the protein. For instance, Fig. 19 shows titration curves at 25 “C for LSZ dissolved in 50 mM KC1 solution obtained by Haynes et al. [ 511 and in 6 M guanidinium chloride {GuHCl) obtained by Tanford and Roxby 11731: the two titration curves differ over the entire pH range, particularly in the acid region. Tanford and Roxby found that LSZ assumes a random-coil (or at least a highly expanded) structure in 6 M GuHCi and that its titration curve in this solvent can be accurately predicted from the intrinsic (i.e. unperturbed) pK, values of the constituent groups. Since the titration curve in 50 mM KC1 differs from that in GuHCl, pK, values for at least some of the residues in the folded conforma-

- notwe state x 6 M GuHCl

-si!i PH

Fig. 19. Proton titration curves at I5’C for hen egg-white lysoqme dissolved in 50 mhl KCI, uhere the native state IS favored, or in 6 hI guanidmium chloride (GuHCIb, where the denatured state is favored.

tion are no longer equal to their intrinsic values. For instance, the p& for glutamic acid residue 35 in native LSZ is 6.3, which is nearly two units above the intrinsic pli, (=4.5) for a free glutamyl carboxyl group. Experiment and theory both suggest that coulomb interactions between charged (titratable) groups on the protein surface are pri- marily responsible for these differences [60,6i.64.173]. For instance, an increase in ionic strength greatly diminishes the difference between the experimental titration curve for a native protein and that calculated on the basis of the intrinsic properties of the constituent groups. Moreover, both Linderstr0m-Lang [ 1741 theory, which evenly distributes the net proton charge over the entire protein surface, and the Tanford-Kirkwood discrete-charge model qualitatively account for the pK, shift and the effect of ionic strength upon it. However, other factors, such as local polarity and involvement of residues in internal hydrogen bonds, may also lead to changes in residue pK,s [ 175.1761. Thus the pK, for a given residue is intimately linked with the residue’s local environment.

The apparent dissociation constant I(, of any one class of dissociating groups (e.g. carboxyl. imidazole etc.) is related to the solution pH by

pK, = pH + log,, [( 1 - x)/%-j (12)

where ptc, is the negative log,, of the apparent dissociation constant and 2 is the degree of dissociation of the groups. K, can also be expressed in terms of the reversible isothermal work AH+G’ required to remove the proton from the (charged) residue to infinity:

In general. proton titration curves for globular proteins are reversible. Thus. at constant temperature and pressure, the Gibbs energy of the protein, G,,. is formally given by

PZ,- / ;r: \

Gpr= J,;- (,g$)T,pdZH+

J ztt+

= -2.303RT PH(G+) G- ( 14) z,+

where G,,(Zi+ is the Gibbs energy at an arbitrary reference state (e.g. pH 7 and 25’C) at which the proton charge on the protein equals ZG+ F per mole [ 1771. The integral of the reciprocal proton titration curve (i.e. pH vs. Z,+) therefore yields G,, as a function of solution pH.

The physical process of removing (or adding) protons provides the dominant contribution to G,,(Z,+); thus G,, is mostly a reflection of changes in the electrical properties (and mass) of the protein. However, G,,(Z,+) will also reflect any changes in protein structure that occur as a result of the titration process. The non-electrostatic Gibbs energy change associated with such a transition

A tranJGnon_e, can also be calculated from proton titration data [ 178,179]:

A Gnon-e, WX”S

= 2.303Z;rr R TX

6 ( p~a-confomation _ pjy p-conformation) dr

Here, ZEr is the total number of ionizable groups in the given class (e.g. carboxyl groups) and 2 and fi represent the two possible conformations. Such transitions are known to occur in r-helix-forming poly(amino acid)s. For instance, Fig. 20 shows pK as a function of r for poly(L-lysine) in aqueous solution (over the pH range 9 cpH < 12). At cc=O, all of the lysine residues carry one unit of positive charge and the poly(amino acid) assumes an expanded coil conformation to minimize the resulting intrachain charge-charge repulsion. At higher pH (and higher G(), the intrachain charge density is reduced to a level where the helix conformation is favored. Figure 20 shows that the coil-to-helix transition occurs when approximately half of the lysine residues are deprotonated (i.e. uncharged). A tranrGnon_e, for this process can be determined from Eq. (15) and the area of the dip in the

Fig. 20. Proton titration data for poly(r_-lysine) in 0.1 M NaBr

solution at ZO’C (-) and adsorbed to negatively charged

polystyrene (u,,= -3.8 pCcm_‘) m 0.1 bl NaBr solution at

20°C (---). Adsorption pH IS 6.0.

dissolved state curve in Fig. 20; it is in the range of -O.lRT to -0.3RT per mole of lysine residue.

In contrast, Bonekamp [ ISO] found no evidence for a coil-to-helix transition in the titration curve for poly(L-lysine) adsorbed (at low surface coverage) to negatively charged PS. On the basis of this and other evidence, he concluded that

- AadsG > - Atrans G, and as a result that the polymer either (a) spontaneously loses (most of) its helical structure upon adsorption or (b) adsorption stabilizes the helix structure to the point where it remains stable under all solution conditions. At higher surface coverages, Bonekamp observed a partial restoration of the helix-to-coil transition, which suggests that the helix conformation is maintained in some of the loops and tails of the adsorbed polymer. Bonekamp’s results imply that structural transitions can occur in adsorbed polyamino acid systems where -A,,,G< - At,,,,G.

Some dissolved state globular proteins also undergo conformational changes as a function of solution pH. For instance, exposure of native rLA to pH values below 4 promotes nearly instantan- eous formation of a conformer (apo state) whose properties differ markedly from those of the native protein [181,182-J. The apo state is characterized

C..4. Haynes und CK Xordr;Colloids Surfuces 8 Bwmtrrjaces I (199-I) 517-566 549

by a loosely packed (but still folded) structure where most internal side-chains have increased rotational freedom with little or no spatial correlation. Transition to this structural inte~ediate appears to be triggered by dissociation of a calcium (Cazf) ion from the high affinity metal binding site on the native protein.

The reciprocal differential titration curve (i.e. - ApH/AZu + vs. -Z,+) for rLA reveals that titration of the protein at pH values below about 6.0 involves only the protonation of its 22 carbox- ylic acid groups. Thus Eq. (15) can be directly applied to proton titration data in the acid region with c( representing the degree of proton dissociation from carboxyl groups. Figure 21(a) shows pK as a function of x for rLA in 50 mM KC1 solution (over the pH range 5 > pH > 2); the wide deep cusp in the curve corresponds to the native- to-apo state transition, for which AtransGnon-cl is estimated to be - 5RT _t 2RT per mole of aLA. This loss in Gibbs energy is of the same order as the stabilization energy -A,uG characterizing the native structure at pH 5 and 25°C; apparently, events in the protein denaturation process which directly influence the magnitude of AN_uG mainly occur in the initial stages of unfolding. Unfortunately, such an analysis is not possible for the many proteins which undergo conformational transitions in the physiological and/or basic pH regions since titrations of different classes of groups overlap at these higher pH values.

Adsorbed state and dissolved state titration

curves for proteins are usually quite different. A striking example involves the titration curve for aLA adsorbed to negatively charged PS in 50 mM KC1 solution. Figure 21(a) ptots plc as a function of r for the adsorbed protein. Two features stand out: (1) the native-to-apo-state transition does not occur in the adsorbed protein; (2) the apparent pK, for carboxyl groups in the adsorbed protein is one to two units higher than that for the dissolved state protein. Both of these features indicate that the surface has a dramatic influence on the properties of the protein. The absence of the native-to-apo-state transition suggests that adsorption causes either (a) spontaneous transition to the apo state, (b) spontaneous transition to a new conformation from which the apo state is no longer accessible, or (c) stabilization of the native structure such that the apo state is no longer favored at low pH. Micro-DSC measurements on xLA adsorbed to negatively charged PS (see Section 8) indicate that the protein most likely undergoes process (b) during adsorption. For instance, temperature scans of adsorbed uLA do not show the characteristic endothermic peak (i.e. AN_& >O) associated with the denaturation of the native state (process (c)t or the apo state (process (a)) respectively.

The shift from 3.2 to 4.3 in the apparent pK, for the carboxyl groups in rLA reveals the strong influence of the charged sorbent on the electrostatic properties of the adsorbed protein. Similar shifts in pK, have been reported for other globular proteins adsorbed to PS [ 183,184]. For instance,

---adsorixd state - - - adsofbcd state -disr&md Slate

Fig. 21. Proton titration data at 3 ‘C for (a) r-lactalbumin and for(b) hen egg-white lysozyme dissolved in 50 mM KC1 and adsorbed to negatively charged polystyrene (a, = - 15.5 PC cm-‘) in 50 mnil KCI. Adsorption pH is 7.0.

the apparent pk’, for carboxyl groups in LSZ increases from 3.0 to 3.9 (see Fig. 2 l( b)). For RNase and HSA adsorption to PS, Norde and Lyklema [184] reported a substantial carboxyl group pk’, shift which increases with increasing negative charge at the polystyrene surface. These results suggest that the average position of protein car- boxy1 groups is relatively close to the sorbent surface. However, dipolemoment data and tertiary structures for LSZ and aLA indicate that different classes of titratable groups are fairly evenly distributed on the surface of each protein [lSl,lSYJ. Moreover, preferential adsorption of deprotonated carboxyl groups would be opposed by their electrostatic repulsion to the negatively charged sorbent. Thus some positively charged residues in the protein must also be located near the sorbent surface.

Figures 22(a) and 22(b) compare dissolved state and adsorbed state titration curves for LSZ and rLA respectively; in both systems, the adsorbed state curve was positioned relative to the dissolved staie curve using the carboxyf group plc, shifts shown in Fig. 21. The two titration curves for each protein differ over a wide pH range. As noted above, these differences are largest in the acid region; however, Figs. 22(a) and 22(b) indicate that the sorbent also affects the titration behavior of at least some of the basic groups on the protein surface. For instance, comparison of titration curves at pH 11 reveals that two basic residues in zLA which titrate in the dissolved state do not titrate in the adsorbed state (over the pH range studied); similarly, adsorption causes three of the

p----T

I I 0 I 1 2 L 6 8 10 I2

basic groups in LSZ to remain protonated. The failure of these positively charged residues to deprotonate at high pH suggests that they are in close contact with negatively charged sulfate groups on the sorbent surface. Similar behavior was reported by Norde and Lyklema [ 1841 for the adsorption of RNase on negatively charged PS.

Based on a sorbent ele~trokinetic charge density of -3.0 PC cm-?, a protein molecular weight of 15 kDa. and a I-P’ of 1 mg m-‘, the formation of two to three basic-residue-suffate-group ion pairs could effectively neutralize al1 of the charge on the sorbent surface in direct contact with an adsorbed protein molecule. In addition, sorbent surface charge can be neutralized by the coadsorption of low molecular weight ions within the adsorbed protein layer. In either case, the distance between any other charged group on the protein and the (neutralized) sorbent surface would only depend on the proximity of the group to those residues involved in ion pair formation and the group’s relative affinity for an apolar environment. Partition coefficients for strong electrolytes in aqueous-organic two-phase systems indicate that the affinity of an ion for an organic phase is related to the ion’s surface charge density and polarizabii- ity: larger ions have lower surface charge densities and higher polarizabilities and thus higher affinities for apolar phases {86]. The apparent enrichment of protein carboxyl groups at the apolar negatively charged PS surface may therefore be related to the large size and high polarizability of the carboxyl ion compared with other charged residues on the protein surface.

Frg. 22. Proton titratEon curves at 25’C for fn) hen egg-white lysozyme and for t b) u-lactalbumin dissolved m 50 mhf KCI and adsorbed to negatlvel) charsed polystyrene (G,= - 15.5 pC cm-’ I in 50 m&I KCI. Adsorption pH IS 7.0.

7.3. Co~~isor~tiu~z of‘fow rnolec~~l~r weight ioi2s (electrophoretic mobilities anti ; potentids)

Adsorption of protein molecules to a charged sorbent surface will involve a redistribution of charge in the interfacial region. Consider, for instance, an adsorption system where the protein and surface carry the same charge sign. Adsorption is then opposed by the global charge-charge repulsion between the protein and the sorbent; the magnitude of the repulsion will increase with decreasing dielectric permittivity in the interfacial layer. However, coadsorption of low molecular weight counterions can substantially reduce this opposing force.

For instance, rLA has a net positive charge at pH 4.0. Comparison of the adsorbed state and dissolved state titration curves for rLA on negatively charged PS at this pH indicates that the average charge on the protein molecule is about 11 units higher in the adsorbed state than in the dissolved state. During the adsorption process, the pH increases to 4.1, which indicates that some of the protons which associate with the adsorbed protein molecules come from the bulk phase (since the system contained no buffer salts). Additional protons come from the dissociation of uncharged carboxyi groups on the surface of dissolved rLA molecules, thereby decreasing the net positive charge on the protein from its equilibrium value at pH 4.0 to its equilibrium value at pH 4.1. Similar behavior has been reported for LSZ (see Fig. X!(a)), RNase, and HSA adsorption to negatively charged PS surfaces [51,184].

The total number of ions incorporated into an adsorbed protein layer can be estimated by comparing the electrokinetic charge density ~,k of the protein,/sorbent complex with the surface charge densities for the bare sorbent and the dissolved protein:

* o = #rotein-sorbent ads ek ek --+Cbent +$?I-A) (16)

where da&&k is the overall change in surface charge density expressed per unit area of sorbent surface, I is the mass of adsorbed protein per unit

area of sorbent, il is the surface area of protein per unit mass, and @rfe” is expressed per unit area of protein surface. However, determination of the three electrokinetic charge densities o~~~~‘*, oirbent and cr~{otF’“-50rbent is seldom straighforward. They are usually obtained from < potentials, which are calculated using particle mobility u data and an appropriate molecular theory; each step in this analysis involves approximations. Nevertheless, a qualitative picture of the composition of the adsorbed layer can be obtained if care and consis- tency are maintained.

Reliable mobility data for the bare sorbent and the protein-covered sorbent can be determined from standard electrophoresis measurements. More-refined electrophoretic techniques, such as moving boundary (Tiselius) electrophoresis, are required to measure dissolved protein mobilities. Figures 23(a) and 33(b) show measured mobilities for the dissolved protein, the bare sorbent, and the protein/sorbent complex in the adsorption system containing negatively charged PS ~~~~~~~ = -3.0 yC cm-‘), 0.05 M KNO,, and RNase or HSA respectively.

The first and main difficulty is to obtain < potentiafs from measured particle mobilities. The parameters { and u are related by a complex set of coupled differential equations which describe the velocity field in the electrolyte solution together with the distribution of ions and electrostatic potential around the charged colloidal particle [ 1861. Analytic solutions to these equations have not been obtained but approximate solutions have been derived for spherical-colloid systems where the thickness of the electrical double layer xv1 is small I: 1873 or large [ 188,189] compared with the particle radius, or when the { potential on the particle is small [ 190,191]. In most cases. however, these simple limiting solutions cannot be applied to mobility data for dissolved proteins or protein/sorbent complexes. For instance, moderate electrolyte concentrations ( 10 mM-0.1 M) and thus intermediate values of w are typically used in protein adsorption experiments and electrophoresis measurements on protein,‘sorbent complexes.

CA. Huynrs and Wl iVordr Colhds Sur$~rs B Bwlnrrrjaces J (IYW) 517-566

Fig. 23. Electrophoretic mobilities at 25 ‘C of bovine pancreas ribonuclease (al and human serum albumin I b) dissolved in 50 mM

KNO, I-_) and adsorbed to negatively charged polystyrene (a,= - 15.5 FC cm -‘) in 50mhI KN03 (---). The electrophoretic

mobility of the bare latex partlcies (...) is also shown.

Determination of < potentials then requires a more advanced theory, such as the non-analytical model of O’Brien and White [186] which accounts for relaxation effects associated with structural per- turbations in the counterion cloud that surrounds the (moving) particle.

The non-spherical shape of many native globular proteins causes additional problems. Approximate relations between < and u are available for cylin- ders, discs and ellipsoids [ 189,192]. However, relaxation effects are absent from all of these theories, and thus they are only applicable at low { potentials. In all cases, calculated [ potentials must be interpreted with caution, particularly when the particle is non-spherical.

Determination of u,~ from < potential data is more exact. The only approximation (and it is a good one) made in relating cek and i is the assumption that Gouy-Chapman theory accurately describes the potential and ion distributions outside the slipping plane of the particle. Then, for a system containing a symmetric electrolyte,

where cel is the electrolyte concentration (mol dmV3) and :e is the charge on both the cation and the anion of the symmetric electrolyte. (The value for z includes the sign of the valency.)

Figure 24 shows calculated &,sG.ek values as a function of solution pH for HSA adsorbed to negatively charged PS, to x-Fe,&, and to posi- tiveIy charged PS in 50 mM KN03. The < potentials were calculated from mobility data using the numerical procedure of O’Brien and White. Thus

Fig. 24. Charge transfer between the solution (includmg protein molecules in the solution) and adsorbed layers of human serum

albumin on various surfaces in 50 mM buffer at X’C.

CA Hawrs and CV, Nwdr,‘Collolds Surfaces 9. Bwinterfucrs 2 ( 1993) 517-366 553

HSA was modeled as a sphere with a diameter equal to 3.82 nm. In general, this is a poor approximation: native HSA has a nearly cylindrical shape in aqueous solution. As a result, such an analysis cannot provide information on the orientation of the adsorbed protein or on the location of adsorbed counter-ions (relative to the protein surface). However, the analysis can provide information on the number of low molecular weight ions incorporated into the adsorbed protein layer (which was our objective).

Interpretation of the Aadsbe_ data in Fig. 24 is facilitated by comparison with plateau adsorption data for HSA on the three sorbent surfaces as a function of pH (see Fig. 13). At low pH (e.g. pH 4), azptein is slightly positive (pl z 4.7 for HSA) and TP’ is essentially the same on all substrates. At this pH, Aadsbelr shifts to more negative values upon changing cfrbent in the positive direction; this trend reflects the transition from preferential adsorption of cations when the sorbent surface is negatively charged to excess incorporation of anions when the sorbent carries a positive charge. At pH 7, HSA is negatively charged and TP’( PS-)<Tp’(r- FezO,) < TP’( PS+). For the negatively charged PS system, for instance, Aadscek is slightly more positive at pH 7 than at pH 4 and I’P’ is substantially lower. Thus the number of cations coadsorbed per HSA molecule increases with increasing negative charge on the protein surface. As expected, the amount of positive charges incorporated (per mole of HSA) decreases with increasing positive charge on the sorbent surfaces. Thus, at pH 7, the negatively charged PS system incorporates the most cations in the interfacial region, while the positively charged PS system takes up the least.

Direct experimental evidence for the coadsorption of low molecular weight ions in adsorbed protein layers is limited. Van Dulm et al. [ 1533 used y-spectroscopy to measure the uptake of Ba’+ ions in the adsorbed protein layer of a system containing HSA, negatively charged PS, and 20 mM BaCll (see Fig. 25); similar results were obtained for MnzC using electron paramagnetic resonance. As predicted by the Aadsrsek analysis. the

c” 1 A

b X 0 *’ A

.- -21 I IX I I I

3 4 5 6 7 8 PH

Fig. 25. Incorporatlon of cations in adsorbed layers of human

serum albumin on negatively charged polystyrene (a,,=

-15.5 FC cm-‘) in 20mM BaCl, solution (x) or m 20mM

MnCl, solution (A.) at 25’C.

number of cations coadsorbed increases with increasing negative charge on the protein surface.

Also shown in Fig. 25 are predicted values for coadsorption of cations in the adsorbed protein layer, based on AL,~s~,k data and the three-layer model of Norde and Lyklema [ 1721. This model, depicted in Fig. 26, assumes complete coverage of the sorbent surface by a compact protein layer. All sorbent surface charge is located at x = 0. The inner region (l), 0 < x < m, contains a fraction of the adsorbed protein charge and any ions trapped between the adsorbed protein molecules and the sorbent surface. The thickness of region 1 is of the order of the diameter of a hydrated ion, which is in the range of a few tenths of a nanometer. The extension of the outer region (3), p < x Gd, is assumed to be comparable to the distance over which charged groups (including their hydration layer) on the protein surface protrude into the aqueous medium; this distance is thought to be about 0.7 nm. Analoguous to the interiors of native-state globular proteins, the central region (2), m < x cp, is considered to be void of isolated charged groups. The thickness of this region follows from measured hydrodynamic thicknesses (see

Fig. 16. Three-layer model for the adsorbed-proteIn~sorbent interface. in which the decay of the electrostatic potenttal is indicated An explanation of the symbols is provided in the text.

Section 6.6) of adsorbed protein layers corrected for the assumed thicknesses of regions 1 and 3.

Because of the requirement of overall electroneutrality,

o,+rr,+0~+CJJ+Gd=O (18)

where the indices refer to the sorbent surface, the three regions of the adsorbed layer, and the diffuse part of the electrical double layer respectively.

Based on the assumptions that b0 is located at x=0, that or, g2 (=O) and cr3 are distributed homogeneously over the regions 1, 2 and 3, and that CT~ is exponentially distributed according to the Gouy-Chapman model [74], Norde and Lyklema [ 1721 derived expressions for (6(-u) across the adsorbed layer and within the bulk aqueous solution. A qualitative representation of #(.x) is shown in Fig. 26.

For all possible values of ciO (derived from titration data for the bare sorbent surface) and (id (= --cJ, &.u) shows a strong dependence on the assumed division of charge between regions I and 3. Since region 1 has a relatively fow dielectric permittivity, any net charge in the contact zone between the sorbent and the protein leads to a large electrostatic potential and is therefore highly unfavorable. Realistically. we would not expect

&s) to attain values larger than a few hundred millivolts. Thus. any mismatch of protein and sorbent charge in region 1 must be compensated by the coadsorption of low molecular weight ions to give a nearly electrically neutral layer. This trend is reflected in the predicted curve in Fig. 25, where the number of ions coadsorbed shows a strong dependence on the electrical states of the protein and sorbent. Here, the number of counterions coadsorbed was estimated from calculated values of Go, which follow from reasonable estimates for 0, (e.g. - 100 mV) and the relation

d$, p-in + ri-p -=- - do, EOEZ ZEOE?;

Equation (19) indicates that 4, is highly sensitive to changes in o-~, and thus to the number of coadsorbed ions.

8. Thermodynamics of protein adsorption

As shown in Eq. ( l), spontaneous adsorption of a protein to an interface is driven by an overall decrease in Gibbs energy (i.e. A,,,G < 0); the affinity of the protein for the interface is reflected in the magnitude of that Gibbs energy reduction. Initial slopes of adsorption isotherms indicate that most globular proteins have high affinities for solid/water interfaces, particularly when the solid is (moderately) hydrophobic {see Section 6). Moreover, as discussed in Section 5, protein adsorption to solid surfaces is usually irreversible. These observations suggest that Aad,G is large (and negative) for most protein adsorption processes.

Accurate determination of AadsG is difficult. The common approach in determining A,,,G by fitting the ascending adsorption isotherm to Langmuir theory (or other reversible-isotherm equations) need not be taken seriously since none of the model conditions (e.g. reversibility, fixed-site adsorption. no conformational changes upon adsorption, no lateral interactions between adsorbed molecules) are met in typical protein- adsorption processes. Instead. A,,,G must be deter-

mined from Eq. ( I) and measured or calculated values for A,& and A,,& Microcalorimetry experiments will provide A&2, but direct measurement of AadoS is not possible. Instead, A.& must be calculated from a knowledge of the many possible conformational and configurational changes that may occur during the adsorption process. Such calculations are now possible for the adsorption of model homopolymers to planar chemically homogeneous solid surfaces (see, for example, the interesting mean-field calculations of Scheutjens and Fleer [ 1931, Cohen Stuart et al. [ 1941. and the Monte Carlo simulations of Cosgrove et al. [ 195]). However, they have not yet been applied to systems containing water molecules, whose configurational properties are dramatically influenced by local chemical environment, nor have they been applied to systems containing proteins, which are heteropolymers havin, 0 remarkably few degrees of rotational and translational freedom in the folded state. Thus the precise determination of A,& (and hence AadsG) remains a distant but very important goal of protein adsorption research.

The A&f data for protein adsorption systems are severely limited despite their obvious importance. Microcalorimetry studies of protein adsorption include those by Haynes and Norde [26] and Arai and Norde [27] for small globular proteins on PS, POM and r-Fe?O, surfaces, by Nyilas et al. [ 1961 for human y-globulin and fibrinogen on silica, and by Norde and Lyklema [33] and Koutsoukos et al. [loll for HSA on PS, AgI and +-Fe,03 surfaces. In each of these studies, Ah,,,H shows complex dependences on system properties such as I-, pH and temperature. For instance, Fig. 27 shows AadsH data at lYp’ for HSA adsorption to various solids [33]; here, A,,,H represents the enthalpy change per square meter of sorbent surface associated with adsorbing an amount of protein equal to l? x A,, where A, is the total surface area of the sorbent. For each surface, AadSH shows a concave-down nearly-parabolic dependence on

Fig. 17. Enthalpy of adsorptton A&f data for human serum albumin on various substrates m 10mM KNO, at 15’C: A, posttively charged polystyrene; A, negatively charged polystyrene (cr,= -2.3 pC cm-‘); i3, negatively charged polystyrene (a, = - 15 5 pC cm -‘): x , sliver iodide sol.

pII. This complex dependence suggests that no single force or interaction dominates the measured heat of adsorption. instead, Aad,H is determined by a balance of energetic subprocesses which occur during adsorption, For instance, A,*,H would reach a minimum near pH 4.7 (i.e. the pI of HSA) if it was dominated by lateral electrostatic interactions between adsorbed protein molecules. Similarly, if global electrostatic forces between the protein and the sorbent surface determine AadsH, we would expect A,Jf to increase monotonically (i.e. become more endothermic) with increasing pH when the sorbent surface is negatively charged and to decrease with increasing pH when the surface is positively charged. As shown in Fig. 27, neither of these trends is observed in typical protein adsorption processes. This is particularly true in the HSA on negatively charged PS and AgI systems where A&$ is negative at high pH despite the combined protein-sorbent and protein-protein electrostatic repulsion.

In contrast, protein-sorbent electrostatic interactions often determine the magnitude of A,&2 for (homo)polyelectrolyte adsorption on charged solid surfaces. For example, calorimetric studies of negativelv charged poly(acry1ic acid) adsorption to titanium oxide [ 1971 indicate that adsorption is exothermic when the two components have

opposite charge signs and endothermic when they have the same charge sign.

Haynes and Norde [ 261 have provided convincing experimental evidence that global electrostatic interactions contribute to Aad,H (see Fig. 28). At low surface coverages. AadsH shifts to more exothermic values as the electrostatic attraction between protein and sorbent increases. The trend is the same at P’ but the magnitude of the change is smaller, which suggests that lateral electrostatic repulsions (and possibly other effects) become stronger at higher surface coverages. The strong dependence of Aads H on T/P* at fairly low surface coverages is surprising since one would expect Aad,H per mole to be constant under conditions where adsorbed protein molecules do not interact.

Apparently. this constant Aad,H per mole regime is confined to very dilute LSZ conditions (i.e. T/P c 0.05 1.

Figure 29 shows A,,,H data for HSA adsorption to hematite at two different surface coverages: T/P’=O.l and r/P’= 1. Lateral interactions between adsorbed protein molecules are likely to be weak at T/l?=O.l and strong at T/l?. Thus subtraction of the low-surface~coverage data from the high gives a crude estimate of the contribution of lateral protein-protein interactions to AadsH. As shown in Fig. 29 (broken curve), lateral interactions are enthalpically favored at pH values near

-rOOOoo i-t rpL

Fig. 28. Enthalpy of adsorption AIL\,& data for hen egg-white lysozyme on negativei) charged polystyrene (go= - 15.5 PC cm-‘) at pH 4 (A). pH 7 (x) and pH 10 (W). Adsorption conditions: 50 mhf KC1 and 25 -C.

Fig. 29. Enthalpy of adsorption A&f data (per mole of adsorbed protein) for human serum albumin on hematite surfaces as a fun&on of pH and surface coverage: *. i-//T*= 0.1: .Z. l-V’= 1.0. The broken curve (---) is the difference between the curves for r;rP’= 1.0 and TIV’=O.~. Adsorption conditions: 10 mM KNO, and 25’C.

the protein’s isoelectric point; in this region, the fairly even distribution of positive and negative charge on the protein surface causes a net electrostatic attraction between neighboring protein molecules. As expected, Iateral interactions become repulsive and endothermic when the protein has a substantial net positive or net negative charge.

The AadsH may also be influenced by a number of other subprocesses, including structural changes in the protein moIecufe (AadsHStr &, (the non- electrostatic contribution to) ion incorporation in the adsorbed layer (AadsHion), changes in the state of sorbent-surface hydration (A,doHhYd), and dissociation of protons from charged residues on the protein surface (A,,,H,-). The contribution to A,,,H of each of these subprocesses can be estimated by assuming that

A,,i,H = AaadsHrtr pr + AhadrH~ + + AadsHion

+ AadsHt,yd + A&% (20)

where dadsHe, represents the electrical contribution to Aad,H due to overlap of electric fields {which includes lateral protein-protein interactions). For instance, Norde and Lyklema [33,198-J used Eq. (10) to obtain a crude estimate of ALiadsHrtr pr (as a function of pH) for the adsorption of HSA from a

0.05 M KNO, solution on negatively charged PS. These results, shown in Fig. 30, are based on measured AadsH values and reasonable estimates of the enthalpy changes associated with the remaining four subprocesses. The data, models and approximations used to calculate AadsHN+,

AadsH,onT AadsHi+ and AzdsHel are well docu- mented by Norde and Lyklema I: 33,198-j and will not be repeated here; instead. we focus on the interpretation of the results.

Norde and Lyklema’s results suggest that the magnitude of A&-I is largely governed by a competition between AadsHstr pr. which is large and endothermic due to the loss of favorable intramolecular interactions within the protein when it adsorbs and unfolds on the sorbent surface, and AadsHion, which is large and exothermic due to the water-water hydrogen bond formation which accompanies the transfer of ions (K’ ions in this case) from water to an apolar environment. At pH 5, for instance, AadsHstc pry 10 mJ m-’ (about 350 kJ per mole protein), which is commensurate with AN_& data for large single-domain proteins in aqueous solution [ 1771, and AadrHion 5 -7RT

Fig. 30. Resolution of enthalpy of adsorption A,,,H (per square meter of sorbent surface) data for human serum albumin on negattvely charged polystyrene (CT”= -1.3 uC cm-‘) in terms of the estimated contributtons of the various adsorption subprocesses: IOIL ion medtum effects: ei. overlap of electric fields; hyd. sorbent surface dehydration effects: H’. proton transfer effects: str pr. changes in protein structure, including hydration.

(about - 17 kJ per moie ion). which is similar to the data of Abraham [ 1991 for the molar enthalpy change associated with transferring a mole of K’ ions from water to various non-aqueous solvents. The value of AadsHstr pr reflects the level of

protein unfolding upon adsorption; it approaches a minimum around pH 4. which is near the isoelec-

tric points of I-ISA (RI ~4.7) and the protein’sorbent complex (PI z 3.9). This trend is

consistent with micro-DSC measurements on small globular proteins which show that protein stabilities (i.e. AN_nG) are usually largest at or near the protein’s pI and gradually fall with increasing net charge on the protein surface (see Section 3.5). Thus the degree of protein unfolding at the solid/liquid interface may be controlled, at least in part, by the native state stability of the protein; when A,_nG is large, AadsHsttr ,,’ is small, and vice versa.

As shown in Fig. 25, ion coadsorption in the interfacial layer is minimum at (or around} the p1 of the protein~sorbent complex. Thus -Aad,H,on is a minimum around pH 4.7 and increases with increasing net charge on the protein molecule and with increasing electrostatic repulsion or attraction between the protein and the negatively charged sorbent surface.

The enthaply changes associated with the remaining subprocesses are predicted to be relatively small. Nevertheless, each makes a significant contribution to the sign and magnitude of AadsH since, at any given pH, AadsHstr Pr and AadsHion largely cancel. The small values for AadsHhyd are consistent with experimental observations that dehydration (at X’C) is driven by a large entropy gain in the water molecules released from the apolar surface (see Fig. 4 and Section 3.1). The value of AadsHH+ is also relatively small, which suggests that the enthalpy change associated with protonating (charged) residues on the surfaces of adsorbed protein molecules is largely cancelled by the concomitant enthalpy change associated with the deprotonation of surface groups of dissolved proteins (see Section 7.3). Similarly, the consis- tently small values predicted for AadsHel suggest

that protein-sorbent and lateral protein-protein electrostatic attractions and:or repulsions are largely eliminated by the coadsorption of low molecular weight charge-compensating ions: this argument is supported by. the strong dependence of ion coadsorption on pH (see Fig. 25).

The heat capacity change upon adsorption, A,&‘,, can be determined by the temperature derivative of A,*& at constant pressure and pH:

As with A,*,H, Aad,Cp can be interpreted in terms of the conformational and configurational changes which occur during the adsorption process

[39,200,201]. Table 13 shows AadsCp values calculated from the AadsH data of Haynes and Norde [26] at 15 and 25’C for the adsorption of zLA and LSZ on negatively charged PS. In both systems, AadsCp shows a strong dependence on solution pH.

Three subprocesses are thought to influence the sign and magnitude of ACiadsCp: ( 1) a loss in heat capacity occurs when hydrophobic surfaces and residues are dehydrated (see Section 3.1); (2) the

Table 13 AadsCp data for lysozyme and for ~-IactaibumiR adsorbed to negatively charsed PS in 50 mM KCI. Values are determined from A_.,,,H data at 15 and X’C and r = I-P’ (data from Haynes and Norde [26])

Sample pH

Lysozyme 4.0 7.0

x-Lactalbumin 3.0 7.0

-LIO - 305 -370

-310 -150 i-120

transfer of ions from aqueous solution to an apolar

environment causes an increase in heat capacity which is proportional to the chaotropic effect of the ion [203]; (3) a loss of ordered secondary

structure in a protein molecule also leads to an increase in heat capacity because of the increased

rotational mobility along the polypeptide chain.

For instance, Brandts [201] estimated that the unfolding of an ordered polypeptide chain (e.g. x-helix or P-sheet) into a random-coil structure

leads to a heat capacity increase of 5-17 J IS-’ per mole of unfolded residues.

Interpretation of the Aad,Cp data in Table 13 in

terms of these subprocesses suggests that sorbent and protein dehydration effects make significant contributions to the protein adsorption process, at least when the sorbent surface is reasonably hydro-

phobic. In the LSZ system, for instance, AadsCp is

negative at each solution pH; similarly, AadrCp is large and negative in the xLA system when the adsorption pH is near the protein’s isoelectric point. For both systems, A,d,C, becomes more positive with increasing charge on the protein molecule. This trend is most pronounced in the

GALA adsorption system, where A,,sC, eventually takes on positive values at high pH. At pH 10.

xLA has a substantial net negative charge, and consequently a relatively low native-state stability (see Section 3.1) and a strong electrostatic repulsion

for the sorbent surface. Thus, compared with adsorption at the protein’s PI, adsorption at pH 10 will probably involve a relatively large change in protein structure and a large transfer of positive

ions from the bulk phase to the interfacial region. Both of these subprocesses (( 2) and (3)) lead to an increase in heat capacity (as shown in Table 13).

As shown in Section 6.1, the surface hydrophobicity of LSZ is greater than that of zLA. Thus.

under otherwise equivalent conditions, additional dehydration of hydrophobic residues on the surface

of LSZ should lead to larger negative values for AadsCp- This is indeed the case: at pH IO, LSZ has a net charge of about -t-4 and AadsCp= -420 uJ m-’ K-r. which is substantially more

C.rl. Ha~nes und W’. Xorde Colhds Surfhces B Biornterfaces 2 ( 19941 517-556 559

negative than the A,,oC, for the rLA system at pH 8.3. Does entropy production drive the adsorption 3, where the protein charge is about + 10. process?

Similar results have been reported by Nor-de [ 203] for the adsorption of HSA on two negatively charged PS surfaces: a low surface-charge-density latex PS( L-), where Q= -2.3 l.tC cme2 and

cddz -b&-w. -?OuCcm-’ in 50 mM KNO,, and a high surface-charge-density latex PS(H-), where oO= - 15.5 uC cm-’ and bd% -0,.,=3.0 pC cmV2. Since the electrokinetic charges on the two surfaces are similar, Norde concluded that the increased surface charge on PS( H-) mainly serves to increase its polarity reiative to PS(L-). Thus, we would expect surface dehydration effects to be larger for adsorption on PS(L-) under otherwise constant conditions. Figures 31(a) and 31(b) show AadsH data at 9’C and 25’C for the adsorption of HSA on PS( L-) and PS(H -) respectively. The importance of sorbent dehydration effects is evident in the PS(L-) system, where A,,,C, is negative at every pH studied. The situation is nearly reversed on the more polar PS(H-) latex, which confirms our expectations and suggests that the contribution of sorbent dehydration effects to Aad,H (and thus to A,&) depends strongly on the polarity of the sorbent and, to a lesser extent, on the surface polarity of the protein.

Protein adsorption to solid surfaces is often endothermic. For instance, the adsorption of rLA on negatively charged PS is endothermic under conditions where the protein and sorbent have the same charge sign. The value of A,,,H is also positive for the adsorption of HSA on positively charged PS (see Fig. 27), RNase on negatively charged PS [33]. and MGB on r-FelO, at pH 9.5 [27]. In each of these systems, spontaneous adsorption must be driven by an increase in entropy (i.e. A,,S>O).

As discussed in Section 3.1, sorbent surface dehydration leads to large positive values for Aa&. The magnitude of the entropy gain which drives sorbent dehydration can be estimated from the the~odynamic properties for the dissolution of small organic molecules in water. For instance, the entropy gain associated with dehydrating PS latex can be estimated from the entropy loss which accompanies the dissolution of ethylbenzene in water; according to NCmethy and Scheraga [204], AS = -91.2 J K-i moi- ’ for the ethylbenzene dissolution process where the first layer of hydration contains approximately 25 mol of water per mole

o,,:-2.3 $ cm-’

-6 I- Fig. 31. Influence of temperature on the enthalpy of adsorption for human serum albumin on negatively charged polystyrene aith (a) low surface charge (uO= -2.3 PC cm-l) and with (b) high surface charge (G,,= - 15.5 $2 cm-‘) in 50 mM KNO3 at 25’C: L.

9’c; +, 25’C.

of ethylbenzene. Thus. the dehydration of I m’ of polystyrene surface. which contains about lOi water molecules before dehydration, leads to an estimated entropy increase of about 60 UJ K-i. Smaller entropy increases will occur during dehydration of protein surfaces since most proteins are amphipolar.

Large positive A,,+$ values can also arise from the unfolding of protein molecules upon adsorption. Creighton [35 3 estimated that the increased rotational freedom of the polypeptide backbone which results from the complete unfolding of a native protein will lead to an entropy gain of 10 to 100 J K-i per mole of amino acid residue. Similar values have been reported by Privalov [52], Brandts [201] and Dill [36]. As discussed in Section 3.4, the relaxation of distorted bond lengths and bond angles within the folded protein molecute may promote a further increase in entropy of about 15 .I IS-’ per mole of unfolded residue [35,.59]. These data suggest that entropy drives protein adsorption in systems where the rotational freedom of the polypeptide backbone is significantly greater in the adsorbed state than in the (native) dissolved state. For instance, consider a loo-residue globular protein where, after adsorption, the average rotational freedom of the polypeptide chain is one fourth that for the fully denatured protein. Based on Creighton’s data, the entropy gain which drives this unfolding process is between 1.4 and 2.9 kJ K-i per mole of protein; therefore, at 300 K, the contribution of -TAadsSEtr pr to A,,,G would be between -420 and -900 kJ per mole of adsorbed protein.

9. A crude estimate of AadsGst, pr

As discussed in Section 8, direct measurement of Aad,G is not possible in most protein adsorption systems. Thus, unlike AadsH, resoIution of A,,,G must be based on reliable estimates of the Gibbs energy changes associated with all of the subprocesses which affect its magnitude:

A,d,G = AadsGrtr pr + AadsGn+ + AadsGion

+ AadsGhyd + AadsGe, (22)

As before, the three-layer model of Norde and Lyklema can be used to estimate AadsGH+, AndsG_

AadsGhyd, and AadsGei. However, determination of the overall Gibbs energy of adsorption also requires an estimate of AadsGstr PT.

Information on the sign of AladsGrtr pr can be gained by calculating the difference quantity

AadsG - A’adsGstr p’ as a function of pH. Figure 32

shows calculated AadsG - AadsGstr pr Values for the adsorption of HSA on negatively charged PS [ 1983. At most pH values, the difference is positive:

thus AadsGst, pr must be more negative than the necessarily negative Value Of A,dsG. A negative value for AadsGEtr pr indicates that structural rearrangements in the protein molecule are thermodynamically favorable and help drive the adsorption process. Unfortunately, the analysis pro\-ides no indication of the magnitude of

AaJstr P’

10. The principle forces involved in protein

adsorption at solid/liquid interfaces

No single force or effect dominates protein adsorption at all solid/water interfaces. However.

as shown in the above case studies. there is now strong evidence that three subprocesses, namely (1) structural rearrangements in the protein molecule, (2) dehydration of (parts of) the sorbent and protein surfaces. and (3) redistribution of charged groups in the interfacial iayer usually make the primary contributions to the overall adsorption behavior. The relative contributions of these subprocesses to the entropy, enthalpy and Gibbs energy of adsorption are shown in Table 14.

The collapse of a polypeptide chain from a large- volume denatured state to a compact native state involves a considerabte loss of confo~ational entropy. Under certain solution conditions, other effects. particularly dehydration of hydrophobic

Fig. 32. Partial resolutton of the Gibbs energy of adsorption AadrG (per square meter of sorbent surface) for human serum

albumin on negatively charged polystyrene (a,=

-2.3 pC cm-a) in 50 mM KNOLI at 75’C.

residues, outweigh this entropic opposition to folding and the native state is marginally preferred. However, there is now substantial evidence that solid/water interfaces upset this delicate balance by providing a region on which the polypeptide backbone can unfold without exposing hydrophobic residues to water molecules. Evidence for rearrangements in protein structure upon adsorp-

tion have come from transmission circular dichroism. NMR and fluorescence spectroscopy, and proton titration data. The most convincing evidence has come from micro-DSC experiments which indicate that most of the ordered secondary structure in native globular proteins is lost when the proteins adsorb to negatively charged polystyrene (a moderately hydrophobic surface).

Additional proof that structural rearrangements in the protein molecule provide a strong driving force for adsorption lies in the general tendency for proteins with low native-state stabilities to adsorb under seemingly unfavorable conditions where. for instance, the surface is hydrophilic and/or the protein and the sorbent surface carry the same charge sign, Examples include the adsorption of zLA and BSA on glass, SiOl and z-Fe203 [27.105]. Here, adsorption cannot be driven by sorbent-surface dehydration or global electrostatic effects: it is therefore likely that conformational entropy production drives the adsorption process in these systems.

Essentially all globular proteins, regardless of their native-state stabilities and electrokinetic charges. adsorb to some extent on hydrophobic

Table 14

Prtmary subprocesses involved in protein adsorption

Subprocess Contrtbution

to A.6.G

Important parameters

Changes m the state of hydration of the sorbent

and the protem surface

Redistribution of charged groups

( 1) Electrical part: overlap of electrtc fields

(2) Chemical part: medium change of

transferred ions

Hydrophobiclty of the sorbent and protein surface

AHSO A.530

Distributton of charge and dielectric constants

before and after adsorptton

Structure of hydratron water: valency and stze

of transferred tons

(C) Rearrangements in the protein structure Structure stabthty of the protein molecule

surfaces. The signature of dehydration effects is a

large decrease in total heat capacity. which is

usually observed in protein adsorption processes

where the sorbent surface is hydrophobic.

Examples include the adsorption of HSA and LSZ

on hydrophobic polystyrene surfaces. Partition

coefficient data for model monomers (e.g. ethylben-

zene) in water-octanol two-phase systems suggest

that dehydration of hydrophobic surfaces results

in an entropy gain of 20-50 PJ K-’ m-’ and a

Gibbs energy reduction of -5 to -20 mJ rn-?,

which alone could easily drive spontaneous protein

adsorption.

The surface properties of the protein molecule

must also affect its adsorption behavior since the

protein surface initially makes the strongest and

closest contact with the sorbent surface. Plateau

adsorption values for similar-size proteins are

strongly dependent on the relative hydrophobicity

of the protein surface; on hydrophobic sorbents.

proteins with high surface hydrophobicities usually

have large TP’ values and little or no tendency to

desorb upon dilution. Moreover, Asakura et al.

[206], Adachi and Asakura [207], and Ohnishi

and Asakura [ZOS] have shown that slight varia-

tions in the amino acid sequence of hemoglobin

make large differences in its surface activity (even

though the variants all have the same molecular

weight).

10.3. Redistribution of charged groups

Plateau values for protein adsorption often show

a maximum at the pI of the protein,isorbent com-

plex. This implies that coulomb interactions influ-

ence adsorption behavior. Protein adsorption

results in a complex (and poorly understood) over-

lap of electric fields which involves charge-charge

interactions between protein and sorbent and

between adjacent protein molecules, ion pairing

between oppositely charged groups on the protein

and sorbent surfaces, redistribution of protons in

the aqueous solution and on the surface of

adsorbed protein molecules, reduction in the

dielectric constant of the interfacial layer. and,

consequently. coadsorption of low molecular

weight counterions to neutralize excess charge in

the interfacial region. Under any solution condi-

tions. a fraction of these subprocesses will favor

adsorption and the remainder will oppose it.

Nevertheless, global electrostatic attractions

appear to drive the adsorption of structurally

stable proteins on charged hydrophilic surfaces.

For instance, lysozyme, which has a relatively high

native-state stability, only adsorbs on z-FezOX

when the surface carries the opposite charge sign.

In contrast, the structurally less stable protein rLA

adsorbs to x-FezO, under all electrostatic condi-

tions [ 273.

10.4. Other effects

Although important, protein size is probably not

a dominant factor in protein adsorption phen-

omena. For instance, hemoglobin (M W z 65 000

D) is far more surface active than fibrinogen

(MW = 330 000 D) despite being only l/5 the size

of fibrinogen [ 2091.

Asymmetric charge distribution on the surface

of a protein can affect its adsorption behavior in

at least two ways. First, oriented protein adsorp-

tion can result from interactions between the

aqueous ‘sorbent interface and a highly charged

“patch” on the protein surface. For instance, the

protein will tend to adsorb patch down if the patch

and the sorbent are oppositely charged. Second.

asymmetric charge distributions will influence a

protein’s permanent dipole moment. Dipole

moments for globular proteins are usually very

large [SS]; z-helices, P-sheets and fixed surface

charges all make substantial contributions to their

values [35]. However, recent total internal reflec-

tion fluorescence data for the adsorption of cyto-

chrome c. which has a very large dipole moment

at most solution pH, on charged silica [210]

suggest that the orientations of the adsorbed mole-

cules are random (i.e. not influenced by the fised

dipole on the protein).

CA. Ha.txrs and Ci: ~Cbrde C&ids Surfims 5 Bwrnrrrfucrs ? ( 199~) 517-566 563

Acknowledgments

This work was funded by a NATO postdoctoral fellowship awarded to C.A.H. by the U.S. National Science Foundation. Thanks are due to Hans Lyklema, Martien Cohen Stuart and Edward Sliwinski for helpful discussions.

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